SELECTIVE INHIBITION OF NF-kappaB ACTIVIATION BY PEPTIDES DESIGNED TO DISRUPT NEMO OLIGOMERIZATION

ABSTRACT

The present invention relates to polypeptides that inhibit the NF-κB signaling pathway and polynucleotides encoding the same. The present invention further provides methods for the modulation of and/or treatment of inflammatory responses, oncogenesis, viral infection; the regulation of cell proliferation and apoptosis; and regulation of B or T lymphocytes in antigenic stimulation, by administering the polypeptides of the present invention to a subject in need thereof. Finally, the present invention provides a method of identifying polypeptides that modulate oligomerization of NEMO.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Application No.60/505,161, filed on Sep. 24, 2003, to U.S. Application No. 60/530,418,filed on Dec. 18, 2003, and to U.S. application Ser. No. 10/948,649,filed Sep. 24, 2004, which are hereby incorporated herein by referencein their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides that inhibit the NF-κBsignaling pathway and polynucleotides encoding the same. The presentinvention further provides methods for the modulation of and/ortreatment of inflammatory responses, oncogenesis, viral infection; theregulation of cell proliferation and apoptosis; and regulation of B or Tlymphocytes in antigenic stimulation, by administering the polypeptidesof the present invention to a subject in need thereof. Finally, thepresent invention provides a method of identifying polypeptides thatmodulate oligomerization of NEMO.

2. Discussion of the Background

Nuclear factor-κB (NF-κB) signaling is an essential signal transductionpathway involved in inflammatory responses, oncogenesis, viralinfection, the regulation of cell proliferation and apoptosis and in thecase of B and T lymphocytes in antigenic stimulation (Ghosh, 1998, Annu.Rev. Immunol.; Karin, 1999, J. Biol. Chem.; Israel, 2000, Trends CellBiol.; Santoro, 2003, EMBO J.). In mammalian cells, there are five NF-κBfamily members that dimerize: RelA, RelB, c-Rel, NF-κB2/p100/p52 andNF-κB1/p105/p50. NF-κB whose predominant form is a heterodimerictranscription factor composed of p50 and RelA subunits, remainssequestered in the cytoplasm through association with members of aninhibitory family of proteins known as IκB. Upon stimulation by thecytokines TNF-α and interleukin-1, endotoxin (LPS), microbial and viralinfections, pro-inflammatory signals converge on the canonical IkBkinase complex (IKK), a protein complex that is composed of two kinasessubunits, IKKα/IKK-1 and IKKβ/IKK-2 and a structural/regulatory subunitNEMO/IKK-γ. Once activated IKK complex phosphorylates IkB proteins,triggering their ubiquitination and subsequent degradation byproteasome. Free NF-κB can then move into nucleus to initiate orup-regulate gene expression. Although IKKα and IKKβ exhibit strikingstructural similarity (52%), exquisite genetic studies have shown thatthey are involved in two pathways for the activation of NF-κB(Pomerantz, 2002, Mol Cell). IKKP is the pro-inflammatory kinase that isresponsible of activation of classical NF-κB complexes whereas IKKα inassociation with NF-κB inducing kinase (NIK) plays essential roles inthe non-canonical NF-κB signaling pathway (Senftleben, 2001, Science).IKKα plays also a role in keratinocyte differentiation but this processis independent of its kinase activity (Hu, 2001, Nature).

The NEMO protein (NF-κB essential modulator) plays a key role in theNF-κB pathway activation. The NEMO protein is associated with IKKα andIKKβ protein kinases in a high molecular weight complex called the IKKcomplex. The IKK kinases are activated by phosphorylation upon anunknown mechanism, which is believed to be a result of NEMOoligomerization (Traincard, 2003, J. Biol. Chem. submitted). Thepresence of the NEMO protein underlies IKK activation sinceNEMO-deficient cells are unable to activate NF-κB in response to manystimuli. NEMO is composed of an N-terminal IKK-binding domain includinga large coiled-coil (CC1). The C-terminal domain functions as theregulatory part of the protein, which has often been reported as abinding template to link many upstream signaling molecules or viralproteins (Ghosh, 1998, Annu. Rev. Immunol.; Santoro, 2003, EMBO J.)Interestingly, mutations responsible for IP and EDA-ID pathologies weremainly found in this part of the molecules (Doffinger, 2001, NatureGen.; Zonana, 2000, Am. J. Hum. Genet.). The C-terminal domain iscomposed of the minimal oligomerization domain including two sucessivescoiled-coil motifs, CC2 (residues 246-286) and LZ (residues 390-412)(Tegethoff, 2003, Mol. Cell. Biol.; Traincard, 2003, J. Biol. Chem.submitted), and a zinc finger motif at the extremity of the C-terminus.

The biochemical mechanisms triggering the activation of IKK in responseto pro-inflammatory stimuli remain unclear. It has been demonstratedthat phosphorylation on two serine residues in the activation T-loopinduces activation of the IKKP. However, the mechanism that leads tothis phosphorylation event is still unknown. One possible mechanismconsists of the conformation change of the kinase induced by NEMOoligomerization (Traincard, 2003, J. Biol. Chem. submitted). This changeof the oligomeric state may induce the T-loop activation by a mechanismof trans-autophosphorylation (Zandi, 1997, Cell; Tang, 2003, J. Biol.Chem.). Consistent with the role of NEMO oligomerization in IKKactivation, mutations in the minimal oligomerization domain failed torescue NF-κB by genetic complementation in NEMO-deficient cellsactivation in responses to many stimuli. Moreover, enforcedoligomerization of NEMO lead to full activation of IKK complex.(Inohara, 2000, J. Biol. Chem.; Poyet, 2000, J. Biol. Chem.; Poyet,2001, J. Biol. Chem.). Recently, the phosphorylation and theubiquitination of NEMO in response to TNF-α have been reported, (Carter,2001, J. Biol. Chem.; Trompouky, 2003, Nature; Kovalenko, 2003, Nature).However, these NEMO modifications have not been demonstrated yet as acrucial step to activate IKK complex in response to severalpro-inflammatory stimuli.

Inhibition of NF-κB activation constitutes a privileged target fordevelopment of new anti-inflammatory and anti-cancer drugs (May, 2000,Science; Poulaki, 2002, Am J. Pathol.). Among many protein actors inNF-κB signaling pathway, IKK complex represents one of the mostpromising molecular targets for discoveries of the new specific NF-κBinhibitors. To minimize the potential toxicity effects in vivo,therapeutical success will greatly depend on the abilities of the NF-κBinhibitors to block activating signals without modifying the basal levelof NF-κB activity. May et al. described a cell-permeable peptidicinhibitor that block specifically the pro-inflammatory NF-κB activationby disrupting the constitutive NEMO interaction with IKK kinases (May,2000, Science; May, 2002, J. Biol. Chem.). Modulating protein-proteininteractions by the rational design of peptide that alter protein'sfunction provides an important tool for both basic research anddevelopment of new classes of therapeutic drugs (Souroujon, 1998, Nat.Biotechnol.), especially with signaling proteins that exhibit flexibleand dynamic binding properties (Pawson, 2003, Science). Numerous studiesof peptide modulators have been described in the literature wherepeptides mediate protein's function by interfering with localization(translocation) (Lin, 1995, J. Biol. Chem.), recruitment to receptor(Chang, 2000, J. Biol. Chem.), intramolecular interactions (Souroujon,1998, Nat. Biotechnol.) and oligomerization (Judice, 1997, P.N.A.S.). Inthe latter, inhibition of HIV-1 gp41 fusion protein with variouspeptides provides a clear proof-of concept (for a review see Chan, 1998,Cell and Eckert, 2001, Ann. Rev. Biochem.).

Under this theory that inhibition of NF-κB activation provides adesirable target for the development of new anti-inflammatory andanti-cancer drugs, the present inventors have set forth to discovercandidate anti-inflammatory and anti-cancer drugs, as well as to providea method of screening for the same.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide polypeptides derivedfrom NEMO that are useful for the regulation and/or inhibition of theNF-κB signaling pathway.

To this end, the present invention provides NEMO-derived polypeptidesthat inhibit the NF-κB signaling pathway.

In one embodiment of the present invention, the NEMO-derived polypeptideis the CC2 domain (murine: SEQ ID NO: 3 or human: SEQ ID NO: 14).

In another embodiment of the present invention, the NEMO-derivedpolypeptide is the LZ domain (murine: SEQ ID NO: 7 or human: SEQ ID NO:16).

In a preferred embodiment of the present invention, the NEMO-derivedpolypeptides are fused via a spacer sequence to a polypeptide having ahigh transduction potential.

Further, in another embodiment of the present invention arepolynucleotides that encode for the NEMO-derived polypeptides eitherwith or without the spacer sequence and the polypeptide having a hightransduction potential.

In yet another embodiment of the present invention is methods ofmodulating or treating disorders regulated by the NF-κB signalingpathway by administering the NEMO-derived polypeptides to a subject inneed thereof. The disorders regulated by the NF-κB signaling pathwayinclude: inflammatory responses, oncogenesis, and viral infection.

The present invention also provides a method of regulating cellproliferation or apoptosis by administering the NEMO-derivedpolypeptides to a subject in need thereof.

In still another embodiment of the present invention is a method ofregulating B or T lymphocytes in antigenic stimulation by administeringthe NEMO-derived polypeptides to a subject in need thereof.

In yet another embodiment, the present invention further provides amethod of identifying polypeptides that modulate oligomerization of NEMOby

a) identifying a candidate polypeptide sequence;

b) creating a polypeptide fusion construct by linking said candidatepolypeptide sequence to a polypeptide having a high transductionpotential via a spacer sequence;

c) contacting a cell culture with the polypeptide fusion construct; and

d) monitoring the activity of the NF-κB signaling pathway;

e) comparing the activity of the NF-κB signaling pathway in the presenceof said polypeptide fusion construct to the activity of the NF-κBsignaling pathway in the absence of said polypeptide fusion construct todetermine the relative inhibition by said polypeptide fusion construct;and

f) correlating relative inhibition by said polypeptide fusion constructto NEMO oligomerization.

The above objects highlight certain aspects of the invention. Additionalobjects, aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following Figures in conjunction with thedetailed description below.

FIG. 1: Functional domains of the NEMO protein.

(A) The murine NEMO protein contains 412 amino acids and multipledomains including the N-terminal IKK binding domain and theoligomerization domain, the proline rich motif (PPP) and the zinc fingermotif (ZF) at the C-terminus. The coiled-coil predictions (open boxes)using the algorithm developed by Wolf et al. (1997, Protein Sci.) andthe NLM conserved motif (black bar) is shown. The sequence ofNEMO₂₅₃₋₃₃₇ (residues 253-337 of SEQ ID NO: 12) corresponding to thesecond coiled-coil (CC2) and leucine zipper (LZ) motifs, which containsall determinants required for NEMO oligomerization (Traincard, 2003, J.Biol. Chem. submitted), is indicated with the NLM conserved motif(residues 293-322 of SEQ ID NO: 12) underlined and with the coiled-coilsequences showed as cylinders below the sequence. Letters immediatelyabove the sequence indicate the heptad repeat ‘a’ and ‘d’ positionswhich is a key feature of coiled-coil sequences (Vinson, 2002, Mol.Cell. Biol.). (B) Multiple sequence alignment of NEMO proteins from Musmusculus (Mm), Homo sapiens (Hs), Bos taurus (Bt) and Drosophilamelanogaster (Dm), showing the NEMO like motif (NLM) shared withNRP/optineurin, ABIN-1/Naf 1, ABIN-2 and ABIN-3/LIND of differentspecies (SEQ ID NOs:19-29, respectively). The multiple sequencealignment was constructed by parsing PSI-BLAST-generated highest-scoringpairs of sequence segments and realigning the same using with CLUSTAL W(Thompson, 1994, N.A.R.). Identical and similar amino acid residues(shaded) are indicated by (!) or (*), respectively.

FIG. 2: Flow cytometry analysis of NEMO peptide uptake

(A) Cellular delivery of NEMO peptides mediated by conjugation with theAntennapedia peptide. 70Z/3 cells were incubated for 2 h at 37° C. inthe absence (W/O) or in the presence of 2 μM BODIPY-tagged Ant-CC2 wildtype (WT), or Ant-CC2 mutant (Mu), or Ant-LZ wild type (WT) or Ant-LZmutant (Mu) peptide as indicated, or with controls corresponding to 2 μMBODIPY-conjugated BSA (BODIPY-BSA) or BODIPY-FL alone. (B) Concentrationdependence of antennapedia-mediated uptake of 0, 0.2, 2 and 20 μMAnt-CC2 at 37° C. for 5 h in 70Z/3-C3 cells (left panel) and FACSkinetic analysis of BODIPY-conjugated Ant-CC2 at 0, 0.5, 1, 2 or 5 hafter addition of 20 μM Ant-CC2 at 37° C.

FIG. 3: Inhibition of LPS-induced NF-κB activation by cell-permeableAnt-CC2 and Ant-LZ peptide

(A) 70Z3 lymphocyte B were stably transfected with pIL1-β-galactosidase,which bears the β-galactosidase gene under the control of the NF-κB (see“Materials and methods”). The resulting cell line, 70Z3-C3, wasincubated for 2 hours in the absence or in the presence of 20 M ofantennapedia peptide (Ant), or BODIPY-labeled antennapedia peptide(BODIPY-Ant), or BODIPY-labeled antennapedia peptide coupled to CC2(BODIPY-Ant-CC2) or LZ (BODIPY-Ant-LZ) peptides, After peptideinternalization, cells were treated for 5 hours with LPS (3 μg/ml, (+)in left panel) or untreated (right panel and (−) in left panel) and theNF-κB activity was measured by β-galactosidase assay. Error barsrepresent the standard deviation of three separate experiments. (B)Concentration dependence of inhibition of LPS-induced NF-κB activationby BODIPY-Ant-CC2 peptide (left panel) or BODIPY-Ant-LZ peptide (rightpanel). Cells were treated as in (A) but with different concentration ofpeptide as indicated. The potential of each peptide to inhibitLPS-induced NF-κB activation was measured by determining the IC₅₀ valuethat correspond to 50% inhibition of LPS-induced NF-κB activation ascompared to the control (no peptide) (C). (D) Effect of the N-fusionsequence of antennapedia on the inhibition of NF-κB activation. Control(no peptide) or CC2, or LZ peptides, with (BODIPY-ANT-CC2,BODIPY-ANT-LZ) or without the antennapedia sequence at the N-terminus(CC2, LZ) were incubated for 2 hours with 70Z3-C3 cells followed with(+) or without (−) the LPS-treatment for 3 hours. NF-κB activity wasthen measured by β-galactosidase assay.

FIG. 4: Specific inhibition of NF-κB activation in response to LPSdepends on a few mutations in the hydrophobic core of CC2 and LZcoiled-coils

Left panels show a helical wheel diagram of CC2 (A) and LZ (B) peptides.The view is from the top the molecule. The (a) through (g) positions,which are an essential feature of coiled coil sequence (Vinson, 2002,Mol. Cell. Biol.) represent sequential positions in each peptidesequence. The first (a) and fourth (d) positions that are generallyoccupied by hydrophobic amino acids constitute the hydrophobic core forparallel as well as antiparallel-coiled coils. Mutations that wereintroduced in (a) positions of the CC2 variant (BODIPY-Ant-CC2 (Mu);shown as residues 6-40 of SEQ ID NO:3, with glycine mutationscorresponding to residues 6-40 of SEQ ID NO:5) or in (d) positions ofthe LZ variant (BODIPY-Ant-(Mu) are shown. In the right panels, 70Z3-C3cells were incubated for 2 hours in the absence (control) or in thepresence of 10 μM of cell permeable wild type (BODIPY-Ant-CC2 (WT)) ormutant (BODIPY-Ant-CC2 (Mu)) CC2 peptides (A) or wild type(BODIPY-Ant-LZ (WT)) or mutant (BODIPY-Ant-(Mu)) LZ peptides (B). Thecells were then extensively washed to remove the excess peptide whichhad not been internalized and the cells were then diluted three timesand allowed to grow for 24 hours before treatment for 5 hours with (+)or without LPS (−). NF-κB activity was measured using theβ-galactosidase assay. Error bars represent the standard deviation oftwo independent experiments.

FIG. 5: Inhibition of NF-κB Activation by the LZ Peptide Occurs Throughthe formation of specific coiled-coil strands.

(A) Sequence alignment of the NEMO-derived LZ (residues 301-336 of SEQID NO: 12) and the GCN4 peptides (residues 23-55 of SEQ ID NO:8). Bothcoiled-coil motifs were aligned using clustalX. Identical and similaramino acid residues (shaded) are indicated by (!) or (*), respectively.(B) Overview and helical wheel diagram of the GCN4 coiled-coil (topview). The amino-acid sequence of GCN4 is shown with its corresponding[a-g] positions and residues that differ from the correspondingNEMO-derived LZ sequence are boxed according to their degree ofconservation. Identical (open square) and similar residues (opentriangle) are indicated. (C) Comparison of the cell permeableNEMO-derived LZ and GCN4 peptide on the inhibition of LPS-induced NF-κBactivation. 70Z3-C3 cells were incubated for 2 hours in the absence (nopeptide) or in the presence of 10 μM of the antennapedia fusion LZ(BODIPY-Ant-LZ) or GCN4 (BODIPY-Ant-GCN4) peptide. Cells were thenextensively washed to remove any peptide excess which was notinternalized, and diluted three times to facilitate 24 hours of growthbefore treatment for 5 hours with (+) or without LPS (−). NF-κB activitywas measured using the b-galactosidase assay. Error bars represent thestandard deviation of two independent experiments.

FIG. 6: Oligomerization properties of NEMO-derived polypeptides with orwithout the antennapedia sequence

All peptides were loaded at a 10 μM concentration on a superdex 75HR10/30 column equilibrated in a buffer containing 0.1 mM DDM to improverecovery (see “Materials and methods”). Chromatographic profiles of theCC2 mutant (dashed line) and the CC2 wild type (solid line) fused(BODIPY-Ant-CC2 (WT), BODIPY-Ant-CC2 (Mu), or not fused to theantennapedia sequence (CC2 (WT), CC2 (Mu)) are shown in left panels, andelution profiles of the LZ mutant (dashed line) and the wild type (solidline) fused (BODIPY-Ant-LZ (WT), BODIPY-Ant-LZ (Mu), or not fused to theantennapedia sequence (LZ (WT), LZ (Mu)) are represented in rightpanels. Elution volumes of globular protein markers are indicated byarrows: Oval, ovalbumin (43 kDa); Chym, chymotrypsinogene A (25 kDa);Ribo, Ribonuclease (13.4 kDa) and Apro, aprotinin (6.5 kDa).

FIG. 7: Association of Ant-CC2 and Ant-LZ peptides to the CC2 peptide

(A) Direct titration of BODIPY-Ant-CC2 (1 μM) with CC2 by fluorescenceanitropy. The concentration of CC2 was determined by amino acidanalysis. Anisotropy values of BODIPY-Ant-CC2 in milliunits (mA) wereplotted against an increasing concentration of the CC2 peptide. Datapoints were fitted (solid line) to the binding isotherm equation with aKD of 15.2 μM (Materials and Methods). The two dashed lines represent astoichiometric titration and intersect at an CC2 concentration of 16 μM.Given the 1 μM concentration of the BODIPY-Ant-CC2, this gives a complexstoechiometrie of 0.8. (B) Direct titration of BODIPY-Ant-LZ (0.1 μM)with CC2 by fluorescence anitropy. The anisotropy values of theBODIPY-Ant-LZ alone (white bar) or in the presence of the CC2 peptide(30 μM, grey bar; 100 μM, black bar) are given in milliunits (mA).

FIG. 8: Cell death induced in the retinoblastoma cell line Y79 byAnt-CC2 and Ant-LZ peptides

Rb cell line Y79 were treated with various concentration of the Ant-CC2(WT) (filled squares) or Ant-CC2 (Mu) (open squares) (A), or Ant-LZ (WT)(filled circles), or Ant-LZ (Mu) (open circles) (B), or Ant peptide(open triangle) (C) for 3 hours (A, B) or 16 hours (C). Cell survivalwas then evaluated using the MTS assay as described in “Materials andmethods”

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined, all technical and scientific terms usedherein have the same meaning as commonly understood by a skilled artisanin enzymology, biochemistry, cellular biology, molecular biology, andthe medical sciences.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

In this application, the present inventors studied the inhibition ofNF-κB activation by peptides designed to disrupt NEMO oligomerization.The present inventors have previously shown that the minimaltrimerization domain comprises the CC2-LZ coiled-coil subdomain and thatthe isolated and/or purified CC2 and LZ domains bind to each other toform a stable trimer of heterodimers. This structural model isreminiscent of the fold of the gp41 ectodomain from HIV-1 (Traincard,2003, J. Biol. Chem. submitted). It consist of a central three-strandedcoiled coil (formed by the CC2 coiled coil motif of NEMO) which issurrounded by the LZ helical motif derived from the C-terminal end ofNEMO, packed in an antiparallel manner around the outside of the CC2coiled-coil. On the basis of this model, the present inventorsrationally designed two cell-permeable peptides corresponding to optimalportions of CC2 or LZ subdomains that mimic the contact area betweenNEMO subunits. Peptide transduction was monitored by FACS and theireffect on LPS-induced NF-κB activation was quantified using a NF-κBdependent β-galactosidase assay in stably transfected pre-B 70Z/3lymphocytes. The present inventors have also demonstrated that the LZpeptide and, to a lesser extent the CC2 peptide, inhibit specificallyNF-κB activation with IC₅₀ values in the μM range. The effects werespecific because control peptides including mutated CC2 and LZ peptidesas well as heterologous coiled-coil peptides (GCN4), had no inhibitoryeffect on NF-κB activation. Furthermore, the present inventors haveshown that these NF-κB peptidic inhibitors induced the cell death in thehuman retinoblastoma cell lines Y79 that exhibit constitutive NF-κBactivity. Collectively, the present inventors have provided a new andpromising strategy to inhibit the NF-κB pathway by targeting NEMO'soligomerization.

The present inventors have proven that NEMO constitutes a preferentialtarget for the search for drugs inhibiting the NF-κB signaling path,because this protein acts upstream from the NF-κB activation path. Therole of NEMO and its various domains was partially studied and publishedin the following article, “NEMO trimerizes through its coiled-coilC-terminal domain.” J Biol Chem, 2002 May 17; 277(20):17464-75. Agou F.et al., a copy of which is incorporated by reference.

In the present invention, the inventors have synthesized peptides thatmimic either the oligomerization domain (CC2 domain=approx. 40residues), or the LZ motif (LZ domain=approx. 40 residues). Thecombination of these peptides alters either the oligomerization of NEMOor the combining thereof with the proteinic effector, in both casesinhibiting the NF-κB pathway.

In an aspect of the present invention, peptide drugs have beenchemically combined with a peptide of 16 amino acids in length(penetratin/antennapedia), thereby enabling intracellular transportthereof possible. The resulting peptides also may be chemically coupledwith a fluorescent tracer in order to monitor internalization into Blymphocyte cell lines through FACS.

The action of these peptides was tested directly on B lymphocytes havingstably integrated the beta-galactosidase carrier gene also bearingupstream from its promoter several NF-κB transcription factor (Clone C3)activation sites (see Examples herein below).

The present inventors have successfully been able to monitor theinhibitory effect of these peptides by measuring the same followingstimulation of the B lymphocytes by LPS.

The results as a whole reveal that the presence of the peptide mimickingthe “CC2” motif reduces the NF-κB activity by 70% as compared with acontrol peptide at a relatively low dose of 20 μM. At thisconcentration, the effect of the “Leucine zipper” peptide is still moresignificant, since its presence in the medium completely eliminates cellresponse.

These new inhibitors of the NF-κB cellular signalling path offer a majoradvantage as anti-inflammatory compounds and also as anti-tumorcompounds, which may be used for the treatment and/or prevention ofcancers and other disorders.

The present invention relates to compounds, peptides, or compositionsthat are used for modulating the oligomerization of NEMO. In particular,the peptide compounds described herein below may be in an isolatedand/or purified or coupled form with or without a vectorizing agent. Itis to be understood that the present invention also embraces peptideshaving at least 70% homology the NEMO-derived polypeptides, so long asthe homologs possess said inhibitory activity. Methods for assessinginhibitory activity of the NEMO-derived polypeptides, and homologsthereof, are provided below and exemplified in the Examples of thepresent application. The peptides of the present invention and the dosesthereof are deemed to possess inhibitory activity when the NF-κBactivity is reduced by at least 50% as compared with a control peptide.

The present invention also relates to pharmaceutical compositionscontaining said peptides, especially for the preparation of medicinesused for the treatment of inflammatory responses, oncogenesis, viralinfection, the regulation of cell proliferation and apoptosis andantigenic stimulation. In a preferred embodiment, the pharmaceuticalcompositions containing said peptides are useful for the treatment ofcancer.

Also embraced by the present invention are methods of obtaining, making,and identifying peptides and compounds that inhibit the NF-κB signalingpathway, in particular by means of the 70Z/3-C3 cell line filed with theCNCM.

As used herein, the term “reduced” or “inhibited” means decreasing theintracellular activity of one or more enzymes in the NF-κB pathwayeither directly or indirectly. The phrase “inhibiting the NF-κB pathway”preferably means that the NF-κB pathway is inhibited by disruption ofNEMO oligomerization.

The term “enhanced” as used herein means increasing the intracellularactivity or concentration of the NEMO derived peptides, which areencoded by the corresponding DNA. Enhancement can be achieved with theaid of various manipulations of the bacterial cell. In order to achieveenhancement, particularly over-expression, the number of copies of thecorresponding gene can be increased, a strong promoter can be used, orthe promoter- and regulation region or the ribosome binding site whichis situated upstream of the structural gene can be mutated. Expressioncassettes which are incorporated upstream of the structural gene act inthe same manner. In addition, it is possible to increase expression byemploying inducible promoters. A gene can also be used which encodes acorresponding enzyme with a high activity. Expression can also beimproved by measures for extending the life of the mRNA. Furthermore,preventing the degradation of the enzyme increases enzyme activity as awhole. Moreover, these measures can optionally be combined in anydesired manner. These and other methods for altering gene activity in aplant are known as described, for example, in Methods in Plant MolecularBiology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, NewYork (1995).

A gene can also be used which encodes a corresponding or variant NEMOderived peptide with a high activity of inhibiting the NF-κB pathway.Preferably the corresponding enzyme has a greater ability than thenative form of the NEMO protein to inhibit the NF-κB pathway, morepreferably at least in the range of 5, 10, 25% or 50% more inhibition.Most preferably the NEMO derived peptides of the present inventionreduce the NF-κB pathway by at least 50%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, as compared to the pathway in thepresence of the native NEMO protein.

In the context of the present Application, a polynucleotide sequence is“homologous” with the sequence according to the invention if at least70%, preferably at least 80%, most preferably at least 90% of its basecomposition and base sequence corresponds to the sequence according tothe invention. According to the invention, a “homologous protein” or“homologous peptide” is to be understood to comprise proteins (peptides)which contain an amino acid sequence at least 70% of which, preferablyat least 80% of which, most preferably at least 90% of which,corresponds to the amino acid sequence of the CC2 region of NEMO (SEQ IDNO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case ofmurine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZregion of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO,wherein corresponds is to be understood to mean that the correspondingamino acids are either identical or are mutually homologous amino acids.It is further to be understood that, as evinced by the Examples of thepresent invention, the homologous peptide of CC2 preferably retains thecoiled-coil motif structure and the homologous peptide of LZ preferablyretains the helical motif structure. With the guidance proffered by theidentification of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 14, and SEQ IDNO: 16 and the detailed description in the Examples below, screening oftheoretical mutations within the scope of the present invention wouldrequire nothing more than a technicians level of skill in the art. Morespecifically, as is routine in the art, with the identification of acandidate sequence (i.e., the regions corresponding to SEQ ID NO: 3, SEQID NO: 7, SEQ ID NO: 14, and SEQ ID NO: 16) the artisan would assay andscreen one or all possible permutations of the said sequence to identifymutants possessing the same or better therapeutic efficacy.

The expression “homologous amino acids” denotes those that havecorresponding properties, particularly with regard to their charge,hydrophobic character, steric properties, etc.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may be determined conventionally by using knownsoftware or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wisconsin Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of identity or similaritybetween two sequences. Gap performs global alignments: all of onesequence with all of another similar sequence using the method ofNeedleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using asequence alignment program such as BestFit, to determine the degree ofsequence homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores. Similarly, when using a programsuch as BestFit to determine sequence identity, similarity or homologybetween two different amino acid sequences, the default settings may beused, or an appropriate scoring matrix, such as blosum45 or blosum80,may be selected to optimize identity, similarity or homology scores.

The present invention also relates to polynucleotides which encode theCC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO:7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ IDNO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case ofhuman-derived NEMO, or fragments thereof, and which can be obtained byscreening by means of the hybridization of a corresponding gene bankwith a probe which contains the sequence of said polynucleotide thatencodes the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO(SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region ofNEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in thecase of human-derived NEMO, or fragments thereof, and isolation of saidDNA sequence.

Polynucleotide sequences according to the invention are suitable ashybridization probes for RNA, cDNA and DNA, in order to isolate thosecDNAs or genes that exhibit a high degree of similarity to the sequencethat encodes the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region ofNEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16)in the case of human-derived NEMO, or fragments thereof.

Polynucleotide sequences according to the invention are also suitable asprimers for polymerase chain reaction (PCR) for the production of DNA,which encodes a NEMO-derived polypeptide having an ability to inhibitthe NF-κB pathway.

Oligonucleotides such as these, which serve as probes or primers, cancontain more than 30, preferably up to 30, more preferably up to 20,most preferably at least 15 successive nucleotides. Oligonucleotideswith a length of at least 40 or 50 nucleotides are also suitable.

The term “isolated and/or purified” means separated from its naturalenvironment.

The term “polynucleotide” refers in general to polyribonucleotides andpolydeoxyribonucleotides, and can denote an unmodified RNA or DNA or amodified RNA or DNA.

The term “polypeptides” is to be understood to mean peptides or proteinsthat contain two or more amino acids that are bound via peptide bonds.

The polypeptides according to invention include polypeptidescorresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ regionof NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16)in the case of human-derived NEMO, or fragments thereof, particularlythose with the biological activity of inhibition the NF-κB pathway, andalso includes those, at least 70% of which, preferably at least 80% ofwhich, are homologous with the polypeptide corresponding to the CC2region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) inthe case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO:14) or the LZ region of NEMO (SEQ ID NO: 16) in the case ofhuman-derived NEMO, or fragments thereof, and most preferably thosewhich exhibit a homology of least 90% to 95% with the polypeptidecorresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ regionof NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16)in the case of human-derived NEMO, or fragments thereof, and which havethe cited activity.

The invention also relates to coding DNA sequences that encode the CC2region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) inthe case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO:14) or the LZ region of NEMO (SEQ ID NO: 16) in the case ofhuman-derived NEMO, or fragments thereof, by degeneration of the geneticcode. One of skill in the art would appreciate that the aforementionedDNA sequences may be based on the full-length DNA sequences for themurine-derived NEMO (SEQ ID NO: 11) and the human-derived NEMO (SEQ IDNO: 17) and thereby these sequences may be used to ascertain the scopeof these sequences in accordance with the present invention.

In the same manner, the invention further relates to DNA sequences thathybridize with DNA sequences that encode the CC2 region of NEMO (SEQ IDNO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case ofmurine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZregion of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, orfragments thereof.

Moreover, one skilled in the art is also aware of conservative aminoacid replacements such as the replacement of glycine by alanine or ofaspartic acid by glutamic acid in proteins as “sense mutations” which donot result in any fundamental change in the activity of the protein,i.e. which are functionally neutral. It is also known that changes atthe N- and/or C-terminus of a protein do not substantially impair thefunction thereof, and may even stabilize said function.

In the same manner, the present invention also relates to DNA sequencesthat hybridize with the DNA sequence that encodes the CC2 region of NEMO(SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case ofmurine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZregion of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, orfragments thereof. The present invention also relates to DNA sequencesthat are produced by polymerase chain reaction (PCR) usingoligonucleotide primers that result from the DNA sequence that encodesthe CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ IDNO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO(SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case ofhuman-derived NEMO, or fragments thereof. Oligonucleotides of this typetypically have a length of at least 15 nucleotides.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing).

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA—DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with approximately 90% identity are sought, the Tm can bedecreased 10° C. Generally, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (Tm); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14,15, or 20° C. lower than the thermal melting point (Tm). Using theequation, hybridization and wash compositions, and desired Tm, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a Tm of less than 45° C.(aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inCurrent Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (2000).

Thus, with the foregoing information, the skilled artisan can identifyand isolated and/or purified polynucleotides, which are substantiallysimilar to the present polynucleotides. In so isolating such apolynucleotide, the polynucleotide can be used as the presentpolynucleotide in, for example, inhibiting the NF-κB pathway.

One embodiment of the present invention is methods of screening forpolynucleotides, which have substantial homology to the polynucleotidesof the present invention, preferably those polynucleotides encoding aprotein having an ability of inhibiting the NF-κB pathway.

The polynucleotide sequences of the present invention can be carried onone or more suitable plasmid vectors, as known in the art for plants orthe like.

In one embodiment, it may be advantageous for propagating thepolynucleotide to carry it in a bacterial or fungal strain with theappropriate vector suitable for the cell type. Common methods ofpropagating polynucleotides and producing proteins in these cell typesare known in the art and are described, for example, in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1982) and Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).

The aforementioned embodiments are described in the context of SEQ IDNO: 3 (CC2 region of the NEMO protein; i.e., amino acids 246-286 of SEQID NO: 12) and SEQ ID NO: 7 (LZ region of the NEMO protein; i.e., aminoacids 390-412 of SEQ ID NO: 12), where SEQ ID NO: 12 is murine-derivedNEMO. The aforementioned embodiments have been further described basedon SEQ ID NO: 14 and SEQ ID NO: 16, which are derived from SEQ ID NO: 18(human-derived NEMO). However, it is understood that the presentinvention preferably provides peptide derivatives of the NEMO proteinthat may be internalized into eukaryotic cells. Internalization of theNEMO peptide derivatives may be imparted by fusing the NEMO peptidederivative(s), or homologues thereof, to a polypeptide having a hightransduction potential. The skilled artisan would readily appreciatethat the term “high transduction potential” as used herein means thatthe polypeptide, and the fusion protein thereof, readily transverses thecellular membrane resulting in the internalization of fusion peptideinto the cellular milieu. Examples of peptides having a hightransduction potential include: the third helix of theAntennapedia/penetratin protein (Ant) (Prochiantz, 2000, Curr. Opin.Cell Biol.), TAT derived peptides (Fawel, 1994, P.N.A.S.), VP22 fromHSV-1 (Stroh C. 2003, Oncogene), Pep. 1 (Morris, 2001, Nature Biotech.).

To exemplify the present invention and the utility thereof, the presentinventors have fused SEQ ID NO: 3 and SEQ ID NO: 7 to theinternalization peptide Ant (SEQ ID NO: 1) separated by a short SKGMQlinker (SEQ ID NO: 9) or by a LKAQADI linker (SEQ ID NO: 10). Theresultant Ant-CC2 construct has the sequence: CRQIKIWFQNRRMKWKKSKGMQLEDLRQQLQQAEEALVAKQELIDKLKEEAEQHKIV (SEQ ID NO: 2), where theN-terminal cysteine has been added for coupling to a fluorophore tofacilitate detection of internalization and/or inhibition. The resultantAnt-LZ construct has the sequence:CRQIKIWFQNRRMKWKKLKAQADIYKADFQAERHAREKLVEKKEYLQEQLEQLQR EFNKL (SEQ IDNO: 6), where the N-terminal cysteine has been added for coupling to afluorophore to facilitate detection of internalization and/orinhibition.

In the present invention the N-terminal cysteine is an optional additionand, as such, this residue may be omitted from the final inhibitorypeptide. Further, in the present invention the linker between thepeptide having a high transduction potential (e.g., Ant) and the CC2 orLZ peptide can be of a variable sequence and/or length, so long as thelinker sequence does not significantly diminish the inhibitory propertyof CC2 or LZ peptide. To this end, the linker may be of a length rangingfrom 1-35 amino acids, preferably 2-25 amino acids, more preferably 3-15amino acids, most preferably 4-10 amino acids. In a particularlypreferred embodiment, the linker sequence is that of SEQ ID NO: 9 or SEQID NO: 10.

As set forth hereinabove, it is to be understood that the homologouspeptide of CC2 preferably retains the coiled-coil motif structure andthe homologous peptide of LZ preferably retains the helical motifstructure, even when in the fusion construct set forth above. As such,the present invention embraces homologous peptides, within the homologyconstraints above, of SEQ ID NO: 2 and SEQ ID NO: 6 (murine-derived) andSEQ ID NO: 13 and SEQ ID NO: 15 (human-derived) with the caveat thatsaid homologous peptides retain the structure of CC2 and LZrespectively, as well as the ability to inhibit the NF-κB pathway.

In an embodiment of the present invention, the inventors explored theN-terminal region of the wild-type NEMO, in particular the NLM conservedmotif (residues 293-322 of SEQ ID NO: 12) appearing in FIG. 1A. To thisend, the following sequences were produced (see Table 1 for thecorresponding sequence):

NLM-DR (SEQ ID NO: 30) Ant.NLM-DR (SEQ ID NO: 31) Tat NLM-DR (SEQ ID NO:32) R7-NLM-DR (SEQ ID NO: 33) R9-NLM-DR (SEQ ID NO: 34)

NLM-DR is a 21 amino acid “motif” (and the corresponding wild type NLMcovering the same amino acid range) derived from the larger 30 aminoacid conserved NLM motif set forth in FIG. 1A. The NLM-DR has beenmutated from the wild type NLM sequence in that the aspartic acid atresidue 111 in the wild type sequence has been replaced by an arginine(see Table 1 and SEQ ID NO: 30). This mutation was selected because, asconfirmed by structural studies, the resulting polypeptide wouldfacilitate an intramolecular salt bridge allowing the stabilization ofthe peptide in its helicoidal form.

From circular dichroism studies (CD), it appears that CC2 and LZpeptides adopt a helicoidal structure, depending on their concentration.CC2 creates a helix more stable than LZ. RMN and Rayon X Diffractionstudies have confirmed the structure of CC2. Always, by CD studies, theNLM-DR peptide is structured as a helix more stable than the wild-typepeptide.

Although, the polypeptides utilized in this example are 21 amino acidslong, it is contemplated in the present invention that the operable sizeof the NLM fragment may be as short as 15 amino acids. In addition, anymutation in the sequence of the NLM polypeptides that are able toreinforce the helicity and the intermolecular interactions between thepeptides and their molecular target would be of particular interest andis within the scope of the present invention.

In the present invention it is speculated that Antennapedia mediatedmonomerization of peptides may be crucial. Specifically, it isspeculated that monomerization allows them to interfer with NEMOoligomerization.

Nuclear factor-kB (NF-κB) signaling is an essential signal transductionpathway involved in inflammatory responses, oncogenesis, viralinfection, the regulation of cell proliferation and apoptosis; and inthe case of B and T lymphocytes in antigenic stimulation (Ghosh, 1998,Annu. Rev. Immunol.; Karin, 1999, J. Biol. Chem.; Israel, 2000, TrendsCell Biol.; Santoro, 2003, EMBO J.). As such, the inventive peptides areuseful for the modulation of and/or treatment of inflammatory responses,oncogenesis, viral infection; the regulation of cell proliferation andapoptosis; and regulation of B or T lymphocytes in antigenicstimulation. Therefore, the present invention provides for a method oftreating the same by administering to a subject in need thereof apeptide in accordance with the present invention.

In the present invention, the “subject in need thereof” may be a human,a domestic animal, a farm animal, or an animal that is generally foundin the wild. For example, the subject may be selected from a human, adog, a cat, a horse, a cow, a mouse, a guinea pig, a sheep, a pig, etc.

Clearly, the amount of the peptide to be administered will depend on thesubject to which it is to be administered. In the case where the subjectis a human, the amount of the peptide to be administered will depend ona number of factors including the age of the patient, the severity ofthe condition and the past medical history of the patient and alwayslies within the sound discretion of the administering physician.Generally, the total daily dose of the compounds of this inventionadministered to a human or other mammal in single or in divided dosescan be in amounts, for example, of from 0.1 mg/Kg/day to 30 mg/Kg/day ofthe peptide, preferably from 0.1 mg/Kg/day to 20 mg/Kg/day of thepeptide, more preferably from 2 mg/Kg/day to 10 mg/Kg/day of thepeptide, in single or multiple doses. Single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.In a preferred embodiment, the preferred dose is 10 mg/patient to beadministered twice a day. In a particularly preferred embodiment, theadministration route is intravenous.

The peptides of the present invention may also be administered as acomponent of a pharmaceutically administrable composition. In otherwords, the peptide may be present in a formulation for administration toa subject in need thereof. The inventive peptide may be the sole activeingredient for NF-κB pathway inhibition or for treatment of inflammatoryresponses, oncogenesis, viral infection, the regulation of cellproliferation and apoptosis and antigenic stimulation. Alternatively,the composition may also contain one or more additional compounds thatmay be used to treat the same. In addition, the peptide of the presentinvention may be in a composition that contains one or more compoundsthat are useful for treatment of a disorder not caused by the NF-κBpathway.

A therapeutically effective amount of the peptides suitable foradministration in the present invention may be administered alone or incombination with one or more pharmaceutically acceptable carriers. Asused herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filer, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil;safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols;such a propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The pharmaceutical compositions suitable for administration in theinvention can be administered to humans and other animals orally,rectally, nasally, parenterally (e.g., intramuscular, intraperitoneal,intravenous or subcutaneous injection, or implant), intracisternally,intravaginally, intraperitoneally, sublingually, topically (e.g., as apowder, ointment, or drop), bucally, as an oral spray, or a nasal spray.The pharmaceutical compositions can be formulated in dosage formsappropriate for each route of administration.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active NEMO-derived polypeptides, the liquiddosage forms may contain inert diluents commonly used in the art. Theinert diluents may include, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. The liquid dosage formfor oral administration may also contain adjuvants, which includewetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Other dosage forms for oraladministration include, for example, aqueous suspensions containing theactive NEMO-derived polypeptides in an aqueous medium in the presence ofa non-toxic suspending agent such as sodium carboxy-methylcellulose, andoily suspensions containing a NEMO-derived polypeptides of the presentinvention in a suitable vegetable oil, for example arachis oil.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of the inventive NEMO-derivedpolypeptides, it is often desirable to slow the absorption of thepeptides from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe NEMO-derived polypeptides then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, dissolving or suspending the drug in an oil vehicleaccomplishes delayed absorption of a parenterally administeredNEMO-derived polypeptides form. Injectable depot forms are made byforming microencapsulated matrices of the NEMO-derived polypeptides inbiodegradable polymers such as polylactide-polyglycolide. Depending uponthe ratio of NEMO-derived polypeptides to polymer and the nature of theparticular polymer employed, the rate of NEMO-derived polypeptidesrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides) Depot injectableformulations are also prepared by entrapping the NEMO-derivedpolypeptides in liposomes or microemulsions which are compatible withbody tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the NEMO-derivedpolypeptides of this invention with suitable non-irritating excipientsor carriers such as cocoa butter, polyethylene glycol or a suppositorywax which are solid at ambient temperature but liquid at bodytemperature and therefore melt in the rectum or vaginal cavity andrelease the peptide.

Solid dosage forms for oral administration include capsules, tablets,pills, prills, powders, and granules. In such solid dosage forms, thepeptide is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier. In addition, the solid dosage form may contain oneor more fillers, extenders, binders, humectants, disintegrating agents,retarding agents, absorption accelerators, wetting agents, absorbents,or lubricants. Examples of suitable fillers or extenders include,starches, lactose, sucrose, glucose, mannitol, and silicic acid, sodiumcitrate and dicalcium phosphate. Examples of suitable binders include,microcrystalline cellulose, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia. Glycerol is an example of asuitable humectant. Examples of suitable disintegrating agents include,agar-agar, calcium carbonate, potato or tapioca starch, maize starch,alginic acid, certain silicates, and sodium carbonate. Paraffin is anexample of a suitable solution-retarding agent. As absorptionaccelerators, any quaternary ammonium compound may be used. Examples ofsuitable wetting agents include, cetyl alcohol and glycerolmonostearate. Examples of suitable absorbents include, kaolin andbentonite clay. Examples of suitable lubricants include, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate. In the case of capsules, tablets and pills, the dosage form mayalso comprise buffering agents.

The tablets may, if desired, be coated using known methods andexcipients that may include enteric coating using for examplehydroxypropylmethylcellulose phthalate. The tablets may be formulated ina manner known to those skilled in the art so as to give a sustainedrelease of the NEMO-derived polypeptides of the present invention. Suchtablets may, if desired, be provided with enteric coatings by knownmethods, for example by the use of cellulose acetate phthalate. They mayoptionally contain opacifying agents and can also be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions that can be used include polymericsubstances and waxes.

Similarly, capsules, for example hard or soft gelatin capsules,containing the active peptide with or without added excipients, may beprepared by known methods and, if desired, provided with entericcoatings in a known manner. The contents of the capsule may beformulated using known methods so as to give sustained release of theactive NEMO-derived polypeptides. In such solid dosage forms the activeNEMO-derived polypeptides may be admixed with at least one inert diluentsuch as sucrose, lactose or starch. Such dosage forms may also comprise,as is normal practice, additional substances other than inert diluents,e.g., tableting lubricants and other tableting aids such a magnesiumstearate and microcrystalline cellulose. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

If desired, the NEMO-derived polypeptides of the present invention canbe incorporated into slow release or targeted delivery systems such aspolymer matrices, liposomes and microspheres. They may be sterilized,for example, by filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can dissolve in sterile water, or some other sterileinjectable medium immediately before use.

The NEMO-derived polypeptides may be formulated into granules with orwithout additional excipients. The granules may be ingested directly bythe patient or they may be added to a suitable liquid carrier (forexample, water) before ingestion. The granules may containdisintegrates, e.g. an effervescent couple formed from an acid and acarbonate or bicarbonate salt to facilitate dispersion in the liquidmedium.

Dosage forms for topical or transdermal administration of the peptide ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. Transdermal patcheshave the added advantage of providing controlled delivery of a peptideto the body. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the peptide in a polymer matrix orgel. The active component is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as may be required. Dissolving or dispensing the peptide in theproper medium can make such dosage forms. Absorption enhancers can alsobe used to increase the flux of the peptide across the skin. Ophthalmicformulation, eardrops, eye ointments, powders and solutions are alsocontemplated as being within the scope of this invention.

Dosage forms for topical administration may comprise a matrix in whichthe pharmacologically NEMO-derived polypeptides of the present inventionare dispersed so that the peptides are held in contact with the skin inorder to administer the peptides transdermally. A suitable transdermalcomposition may be prepared by mixing the pharmaceutically activeNEMO-derived polypeptides with a topical vehicle, such as animal andvegetable fats, oils, petrolatum, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof, together with apotential transdermal accelerant such as dimethyl sulphoxide orpropylene glycol. Alternatively the active NEMO-derived polypeptides maybe dispersed in a pharmaceutically acceptable paste, cream, gel orointment base. The amount of active NEMO-derived polypeptides containedin a topical formulation should be such that a therapeutically effectiveamount of the peptides are delivered during the period of time for whichthe topical formulation is intended to be on the skin.

Powders and sprays can contain, in addition to the NEMO-derivedpolypeptides of this invention, excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants such as chlorofluorohydrocarbons. Thetherapeutically active NEMO-derived polypeptides may be formulated intoa composition, which is dispersed as an aerosol into the patient's oralor nasal cavity. Such aerosols may be administered from a pump pack orfrom a pressurized pack containing a volatile propellant.

The therapeutically active NEMO-derived polypeptides used in the methodof the present invention may also be administered by continuous infusioneither from an external source, for example by intravenous infusion orfrom a source of the NEMO-derived polypeptides placed within the body.Internal sources include implanted reservoirs containing theNEMO-derived polypeptides to be infused which is continuously releasedfor example by osmosis and implants which may be (a) liquid such as anoily suspension of the peptides to be infused for example in the form ofa very sparingly water-soluble derivative such as a dodecanoate salt ora lipophilic ester or (b) solid in the form of an implanted support, forexample of a synthetic resin or waxy material, for the NEMO-derivedpolypeptides to be infused. The support may be a single body containingthe entire quantity of the peptides or a series of several bodies eachcontaining part of the quantity of the peptides to be delivered. Theamount of active peptides present in an internal source should be suchthat a therapeutically effective amount of the peptides are deliveredover a long period of time.

The present invention further provides a method of identifyingpolypeptides that modulate oligomerization of NEMO by

a) identifying a candidate polypeptide sequence;

b) creating a polypeptide fusion construct by linking said candidatepolypeptide sequence to a polypeptide having a high transductionpotential via a spacer sequence;

c) contacting a cell culture with the polypeptide fusion construct; and

d) monitoring the activity of the NF-κB signaling pathway;

e) comparing the activity of the NF-κB signaling pathway in the presenceof said polypeptide fusion construct to the activity of the NF-κBsignaling pathway in the absence of said polypeptide fusion construct todetermine the relative inhibition by said polypeptide fusion construct;and

f) correlating relative inhibition by said polypeptide fusion constructto NEMO oligomerization.

In this method, the candidate polypeptide sequence preferably has acoiled-coil or helical structure. More preferably, the candidatepolypeptide sequence has 20-60 amino acids. It is also preferred thatthe candidate polypeptide sequence be derived from NEMO.

As stated in the embodiments above, the spacer sequence may have alength ranging from 1-35 amino acids, but shorter lengths may also beemployed (supra). Examples of the spacer sequence includes: SEQ ID NO: 9and SEQ ID NO: 10. Additionally, an example of the polypeptide having ahigh transduction potential is a polypeptide having the amino acidsequence of SEQ ID NO: 1.

In a preferred embodiment, the cell culture contains pre-B 70Z/3lymphocytes that have been transfected with NF-κB dependentβ-glactosidase reporter gene.

In order to ensure that the polypeptide fusion construct is actuallyincorporated into the cells contained in the cell culture it is desiredthat the polypeptide fusion construct have an N-terminal cysteineresidue. In this manner, the polypeptide fusion construction may belabeled by chemically reacting the cysteine residue with a fluorophore(e.g., BODIPY) thus enabling monitoring of cellular uptake by atechnique such as FACS.

Accordingly, the method of identifying polypeptides that modulateoligomerization of NEMO may also include the following steps:

b-1) labeling said polypeptide fusion construct; and

c-1) monitoring cellular uptake of the labeled polypeptide fusionconstruct.

Further, one of skill in the art may also be able to correlate NF-κBpathway inhibition with modulation of NEMO oligomerization by a pulldown experiment with tagged peptides to show that NEMO associates invivo with the peptides. The oligomeric state of this peptide associatedNEMO protein could be characterized (cross-linking, gel filtration). Invitro, Inhibition of the anisotropy increase resulting from theassociation of fluorescent antennapedia labelled CC2 or LZ peptides withCC2 or LZ peptides, mimicking NEMO oligomerization, could be used totest compounds inhibiting NF-κB pathway in vivo.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Materials and Methods

Cell Culture, Stable Transfections and Cell Lines

The grow conditions of the murine pre-B 70Z/3 were as described inCourtois et al., 1997 Mol. cell. Biol. 70Z3-C3 stable cell lines wereprepared by electroporation as described in Courtois et al. with theplasmid cx121acZ-kB (a kind gift from G.R. Crabtree), bearing threetandem copies of NF-κB sites in the IL-2 promoter (Fiering et al.,1990), The human retinoblastoma cell lines Y79 were purchased from theAmerican Type Culture Collection (Manassas, Va.) and grown in RPMI 1640medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin,and 10% fetal calf serum (FCS).

FACS Analysis

0.5×10⁶ 70Z/3-C3 cells in 0.5 ml were incubated at 37° C. for differenttimes and with various concentrations of peptides as indicated in theFigure legends. The cell suspension was centrifugated at 1,000×g at roomtemperature and the cell pellet was then washed three times with PBSbuffer (1 ml), and finally was resuspended with 500 μl of PBS buffercontaining 0.1% sodium azide. Fluorescence analysis was performed with aFACSCalibur (BD Biosciences) and a minimum of 15,000 events per samplewas selected. All experiments were performed in duplicates.

Peptide Synthesis and Purification

Peptides were synthesized as described in Mousson et al. 2002(Biochemistry, 41 p13611-p13616), by using continuous-flow Fmoc/tBuchemistry (Chan, WC, and White, P. D. (2000); Fmoc Solid Phase PeptideSynthesis. A pratical approach) on an Applied Biosystems (Foster City,Calif.) Pioneer peptide synthesiser. All chemical reagents werepurchased from Applied Biosystems. All peptides were blocked at theN-terminus with an acetyl group and at the C-terminus with an amide. Asingle extra-cysteine residue was incorporated at the N-terminus of thepeptides for subsequent specific labeling (see Table 1).

TABLE 1 Sequence of NEMO derived peptides Theoretical ExperimentalConstructions mass mass with NEMO Name Sequence¹ (Da) (Da) Humansequences BODIPY- B-CRQIKIWFQNRRMKWKK 2805.19  2805.06 ± 0.52 Ant (SEQID NO: 1) BODIPY- B- 7433.01  7433.33 ± 0.46 B- Ant-CC2CRQIKIWFQNRRMKWKKSKGMQLEDLRQ CRQIKIWFQNRRMKWKKSKGMQLEDLK (WT) QLQQ QQLQAEEALVAKQELIDKLKEEAEQHKIV QAEEALVAKQEVIDKLKEEAEQHKIV (SEQ ID NO: 2) (SEQID NO: 13) CC2 (WT) SKGMQLEDLRQQLQQAEEALVAKQELIDKL 4155.75  4155.86± 0.53 SKGMQLEDLKQQLQQAEEALVAKQEVIDK KEEAEQHKIV LKEEAEQHKIV (SEQ ID NO:3) (SEQ ID NO: 14) BODIPY- B- 7265.18  7265.04 ± 0.35 Ant-CC2CRQIKIWFQNRRMKWKKSKGMQLEDLRQ (Mu) Q G QQ AEEA G VAKQEL G DKLKEEAEQHKIV(SEQ ID NO: 4) CC2 (Mu) SKGMQLEDLRQQ G QQAEEA G VAKQEL G DK 3987.43 3987.43 ± 0.55 LKEEAEQHKIV (SEQ ID NO: 5) BODIPY- B- 8064.2    8063.9± 0.48 B- Ant-LZ CRQIKIWFQNRRMKWKKLKAQADIYKADCRQIKIWFQNRRMKWKKLKAQADIYKAD (WT) FQAE FQAE RHAREKLVEKKEYLQEQLEQLQREFNKLQAREKLAEKKELLQEQLEQLQREYSKL (SEQ ID NO: 6) (SEQ ID NO: 15) LZ (WT)LKAQADIYKADFQAERHAREKLVEKKEYLQ 5318.08   5318.21 ± 0.5 LKAQADIYKADFQAERQAREKLAEKKELL EQLEQLQREFNKL QEQLEQLQREYSKL (SEQ ID NO:7) (SEQ ID NO: 16) BODIPY- B- 8012.04  8011.98 ± 0.26 Ant-LZCRQIKIWFQNRRMKWKKLKAQADIYKAD (Mu) FQAE RHAREKLVEKKEY S QEQLEQ S QREFNKL(SEQ ID NO: 8) LZ (Mu) LKAQADIYKADFQAERHAREKLVEKKEY S Q 5265.92  5268.82± 0.18 EQLEQ S QREFNKL (SEQ ID NO: 9) BODIPY- B- 7315.48  7314.76 ± 0.40Ant-GCN4 CRQIKIWFQNRRMKWKKSKGMQRMKQ (WT) LEDK VEELLSKNYHLENEVARLKKLVGER(SEQ ID NO: 10) NLM-DR LKAQADIYKA R FQAERHAREK 2570.94 2570.78 +/− 0.21LKAQADIYKA R FQAERQAREK (SEQ ID NO: 30) (SEQ ID NO: 35) BODIPY- B-5317.08 5316.38 +/− 0.26 B- Ant-NLM- CRQIKIWFQNRRMKWKKLKAQADIYKA RCRQIKIWFQNRRMKWKKLKAQADIYKA R DR FQAE FQAE RHAREK RQAREK (SEQ ID NO: 31)(SEQ ID NO: 36) BODIPY- B- 4630.17 4630.11 +/− 0.22 B- TAT-NLM-CYGRKKRRQRRRLKAQADIYKA R FQAERH CYGRKKRRQRRRLKAQADIYKA R FQAER DR AREKQAR (SEQ ID NO: 32) EK (SEQ ID NO: 37) BODIPY- B- 4181.65 4181.60+/− 0.69 B- R7-NLM- CRRRRRRRLKAQADIYKA R FQAERHAREK CRRRRRRRLKAQADIYKA RFQAERQAREK DR (SEQ ID NO: 33) (SEQ ID NO: 38) BODIPY- B- 4494.02 4493.81+/− 0.23 B- R9-NLM- CRRRRRRRRRLKAQADIYKA R FQAERHAR CRRRRRRRRRLKAQADIYKAR FQAERQAR DR EK EK (SEQ ID NO: 34) (SEQ ID NO: 39) (1) In all peptidesthe N-terminus contains a cysteine residue for convenience of specificpeptide coupling with the maleimide group as described in the “Materialand Methods.” The sequences of antennapedia, TAT and poly-arginine (R7or R9) fused to the NEMO sequence (plain text) are highlighted in boldcharacters. Residues that may be involved in coiled-coil sequence areunderlined and those that were replaced in the CC2 and LZ mutants and inNLM-DR are underlinedin bold characters. B = Bodipy (The C-terminalBodipy modification has been omitted from the sequences appearing in theSequence Listing) Within the scope of the present invention it iscontemplated that Bodipy and/or the N-terminal cysteine may be removedand used as described herein.

Crude peptides were directly purified by reverse-phase medium-pressureliquid chromatography (MPLC) on a NUCLEOPREP™ 20 μM C18 100 Åpreparative column, using a linear gradient of acetonitrile (1%/min) in0.08% aqueous trifluoroacetic acid (TFA) (pH 2) for 60 min at a flowrate of 18 ml/min. The purity of the peptides was verified on anucleosil 5 μM C18 300 Å analytical column, using a linear gradient ofacetonitrile (0.5%/min) in 0.08% aqueous TFA (pH2) for 20 min at a 1ml/min flow rate. Conjugation of the Fluorophore Bodipy® FL N-(2aminoethyl)maleimide (Molecular Probes) to the sulfhydryl group wasunder equimolar conditions at pH 6 in 50 mM ammonium acetate buffer for30 min in the dark. The mixture was then loaded on a NUCLEOPREP™20 μMC18 100 Å preparative column to purify the BODIPY conjugated peptide. Incell death experiments, all peptides devoid of BODIPY-labeling weresubjected to treatment with iodoacetamide to prevent any oxidation ofcysteine residue. All purified peptides were then quantified by aminoacid analysis and finally characterized by using positive ionelectrospray ionization mass spectrometry (ES+). Once integrity of thepeptides and coupling efficiency were verified by mass spectrometry, theextinction coefficients of the peptides were measured at 505 nm or at280 nm when peptides contained aromatic residues (see Table 1).Stability of the labeling was monitored periodically by measuring theabsorbance of peptides at 280 nm and at 505 nm and by calculating theabsorbance ratio. All peptides were dissolved in water to stocks of 2mM.

NF-κB Inhibition Assays

In a first procedure 2.2×10⁵ 70Z/3-C3 cells in 220 μl of RPMI 1640supplemented with 10% fetal calf serum (FCS) and 50 μM β-mercaptoethanolwere placed in a 96-well plate and incubated with various concentrationsof peptide (0 to 20 μM) at 37° C. in 5% CO₂ incubator. After two hoursan equal portion (100 μl) of each cell sample transferred in two wellscontaining each 10⁵ cells. One aliquot of cells was then treated for 5hours with lipopolysaccharides from Salmonella abortus (Sigma) at 0.5μg/ml final concentration, and the other one left-treated. After 5hours, cells were centrifuged at 400×g for 5 min at room temperature andthe cell pellets were washed three times with cold PBS (250 μl) bycentrifugation. Cells were then lysed in the lysis buffer (25 mMtris-phosphate buffer at pH 7.8 containing 8 mM magnesium chloride, 1 mMdithioerythreitol, 1% Triton X-100, 15% glycerol and a proteaseinhibitor mixture (Roche)), and samples were centrifuged at 4° C. for 20min to clarify the lysate. The supernatant was then kept on ice, and 30μl was then assayed to measure the β-galactosidase activity with a plateluminometer (Berthold) using the GALACTON-STAR™ as chemiluminescentsubstrate (BD Biosciences Clontech, Bronstein et al., 1989). Backgroundof reaction was measured by mixing for 1 hour 30 μl of lysis buffer withthe reaction buffer (196 μl) and the GALACTON-STAR™ substrate (4 μl)provided by BD Biosciences. In a second procedure and a more stringentassay, 70Z/3-C3 cells (2.2×105 in 220 μl medium) were centrifuged at400×g at room temperature after peptide internalization for 2 hours, andcell pellets were washed three times with 200 μl of PBS bycentrifugation. Cells were then diluted three times with completemedium, and allowed to grow for at least 24 hours. The following stepsare identical to the first procedure.

Cell Death Assays

The detection of cell death was performed using the MTS assay providedby Promega (CellTiter 96® AQueous one solution cell proliferationassay). Briefly, 0.3×10⁶ Y79 cells in 450 μl were treated with 50 μl ofthe wild type Ant-CC2 and Ant-LZ peptides (0.1 to 20 μM) or theirmutants Ant-CC2 (Mu), Ant-LZ (Mu) or the Ant or left untreated inserum-free RPMI medium at 37° C. After an incubation of 1 or 14 hours,an aliquot of the cell suspension (200 μl, 0.12×10⁶ cells), was thentransferee in 96-well plates and mixed with the MTS solution (40 μl)containing the MTS compound and the phenazine ethosulfate. Two hoursafter, the quantity of formazan produced by viable cells was measuredusing an automated microplate reader (Bio-TeK Instruments, INC) at 490nm absorbance. Cell survival was observed under microscope and wasestimated as a percentage of the value of untreated controls. Thebackground of the reaction was determined by mixing the MTS solutionwith cell-free RPMI medium. To increase the sensitivity of the celldeath assay, the present inventors used peptides devoid ofBODIPY-labeling because the absorption spectra of the fluorophoroverlaps with that of the formazan product. All experiments wererepeated twice and each experiment condition was repeated in duplicatewells in each experiment.

Analytical Gel Filtration Experiments

The oligomeric states of peptides were determined by filtration asdescribed in Traincard et al., 2003. In brief, 500 μl samples wereloaded on a SUPERDEX™ 75 HR 10/30 column equilibrated in 50 mM Tris-HClpH 8.0 containing 200 mM NaCl and 0.1 mM DDM, at a constant flow rate of0.4 ml/min. The presence of the DDM detergent was added in theequilibrium buffer to minimize the adsorption in the column and toincrease the peptide recovery. The column was calibrated in the sameequilibrium buffer with blue dextran 2000 (void volume),dithioerythritol (total volume), bovine serum albumin (67 kDa, Rs=35.2Å), ovalbumine (43 kDa, Rs=27.5 Å), chymotrypsinogen A (25 kDa, Rs=21.1Å), ribonuclease A (13.7 kDa, Rs=16.4 Å), cytochrome C (12.4 kDa,Rs=17.7 Å) and aprotinin (6.5 KDa, Rs=13.5 Å).

Fluorescence Anisotropy Measurements

Anisotropy measurements were performed with a PTI QUANTAMASTER™fluorometer equipped with polarizers for the excitation and emissionbeams. This instrument uses a PMT in the L-configuration. Allexperiments were carried out in a 1 cm path-length cuvette at 22° C.with excitation and emission wavelengths at 495 nm and 520 nm,respectively. The bandpass of excitation and emission monochromators wasset at 2 and 4 nm, respectively. Steady-state fluorescence anisotropywas expressed as millianisotropy (mA) and was calculated according tothe equations: (1) A=(I_(VV)−GI_(VH))/(I_(VV)+2GI_(VH)); (2)G=I_(HV)/I_(HH).; where A is anisotropy, G is a correction factor forwavelength-dependent distortion and I is the fluorescence intensitycomponent (subscript referring to the vertical and horizontalpositioning at the excitation and emission polarizers, respectively).Experiments were at least performed twice and each data is the result of20 records along a 2 min period. All measurements were carried out in 50mM Tris-HCl buffer at pH 8 containing 150 mM KCl. The present inventorsverified that at the BODIPY-Ant-CC2 and BODIPY-Ant LZ concentration used(1 μM and 0.1 M respectively), the filter effect was negligible. TheBODIPY-Ant-CC2 peptide was preincubated overnight at 22° C. alone orwith increasing concentrations (1-125 μM) of CC2 prior to anisotropymeasurement. The BODIPY-Ant-LZ peptide (100 nM) was preincubatedovernight at 22° C. alone or with 10 μM and 100 μM concentrations of CC2(see legend FIG. 7) prior to anisotropy measurement. The dissociationconstant parameter was estimated by globally fitting the anisotropy datato binding isotherm equation as described in Agou et al. (J Biol Chem.2004 Jul. 2; 279(27):27861-9) using KALEIDAGRAPH™ nonlinear regressionsoftware (Synergy Software, reading PA). The binding stoichiometry, n,was estimated from the intersection of lines (dashed lines in FIG. 7)drawn through the descending and plateau region of the anisotropy data.

Results

Rational Design of NEMO Derived Peptides that Block NF-κB Activation

The present inventors previously demonstrated that the minimallytrimerization domain of NEMO comprised of the sequence 251 to 337 (FIG.1A). This region likely contains two coiled-coil sequences of about 35residues denoted CC2 (residue 253-285) and LZ (301-337) at the N- andC-terminus respectively. Although the structure of the minimaloligomerization domain has not yet been determined, several biochemicalstudies combined with the fluorescence polarization method prompted usto propose that the CC2/LZ trimer probably forms a six-stranded helicalbundle composed of closely packed CC2 and LZ coiled-coils in anantiparallel orientation (Traincard, 2003, submitted). FurthermorePSI-BLAST searches reveal that this domain of NEMO contains a conservedmotif of 20 residues called “NEMO like Motif” (NLM) which is shared withfour other proteins including ABIN-1 (Heyninck, 1999, J. Cell. Biol.),ABIN-2/NAF (Van Huffel, 2001, J. Biol. Chem.), ABIN-3/LIND (Staege,2001, Immunogenetics) and NRP/optineurin (Schwamborn, 2000, J. Biol.Chem.) (FIG. 1B). Interestingly, most of these proteins including theconserved motif of ABIN-1 (Heyninck, 2003, FEBS Letters), the C-terminaldomain of NEMO (Le Page, 2001, Virology) and ABIN-2 (Liu WK, 2003,Biochemical Journal) or ABIN-3/LIND (Heyninck, 2003, FEBS letters)proteins have been shown to inhibit NF-κB activation in adominant-negative manner when overexpressed in cells. Two additionalreferences that disclose ABIN peptides interacting with NF-κB alsowarrant mention: WO 99/57133 and WO 03/00280.

Since disrupting NEMO oligomerization represents a potential therapeuticstrategy for inhibiting NF-κB activation, the present inventors designedNEMO-derived partner peptides that mimic either the CC2 or the LZsequence (Table 1). It is interesting to note that, unlike the CC2peptide, the LZ peptide also includes the NLM motif at the N-terminalextremity. To mediate all peptide uptake into cells, the presentinventors conjugated a functional analogue at the peptide N-terminuscomprised of the 16-amino acid sequence derived from the third helix ofthe Antennapedia/penetratin protein (Ant). This amphipatic helix acts asan internalization vector (Prochiantz, 2000, Curr. Opin. Cell Biol.).Most of antennapedia fusion peptides were labeled with the BODIPYfluorophore to analyze the transduction potential of each peptide intothe cells. Specific labeling was performed by adding a single cysteinresidue at the extremity of the N-terminus and sequence integrity wasverified by mass spectrometry (see “Materials and methods” and Table 1).

Cellular Uptake of NEMO Derived-Peptides Mediated by the AntennapediaFusion Peptide

The uptake of BODIPY labeled NEMO peptides into living cells weremonitored by fluorescence activated cell sorting (FACS) which is aconventional tool used to quantify cellular internalization. FIG. 2Ashows FACS analyses of cells treated with Ant-CC2 (WT), Ant-CC2 (Mu),Ant-LZ (WT) or Ant-LZ (Mu) BODIPY-peptides for 2 h at 37° C., and werecompared with those of the autofluorescence of untreated cells andcontrol cells treated with an equal concentration of free BODIPY or withBODIPY-conjugated BSA. Consistent with the role of antennapedia peptideto transduce peptides and proteins into mammalian cells, 100% of70Z3-C3-cell line was similarly transduced by the four different NEMOpeptides, suggesting that all of the cells in the treated populationhave a near identical intracellular concentration of NEMO-derivedBODIPY-peptides. Comparative analysis indicate that untreated cells andtreated cells with BODIPY-BSA or free BODIPY exhibit a similar cellfluorescence, verifying that our extensive washing protocol before FACSanalysis was optimal to minimize any contribution of surface-boundpeptide in measuring NEMO peptide internalization (see “Materials andmethods”). Thus, these data suggest that the observed cellularfluorescence signaling mostly reflects the intracellular concentrationof transduced NEMO peptide and not a non-specific adsorption onto themembrane surface.

Then present inventors next investigated the kinetic and concentrationdependency of cellular uptake for the Ant-CC2 BODIPY peptide keeping inmind that the transduction of other NEMO peptides should occur in asimilar fashion (FIGS. 2B and 2C). FACS analysis 5 h after addition of70Z3-C3 cell treated with 0.2, 2 or 20 μM BODIPY-Ant-CC2 peptide at 37°C. demonstrate the linear dependancy of the intracellular concentrationas a function of the incubated concentration of the antennapedia fusionpeptide as widely reported in literature (Lindsay, 2002, Current Opinionin Pharmacology). Notably, the cells treated with the Ant-CC2 at 20 μMand at 37° C. already reach maximum intracellular concentration in 30min and remain unchanged for up to 5 h. Since the time to induce astrong NF-κB activation in response to LPS requires 3-5 hours of celltreatment, these results indicate that the intracellular concentrationof each peptide remains constant during the LPS stimulation.

Specific Inhibition of LPS-Induced NF-κB Activation by Cell PermeableCC2 and LZ

To analyze the inhibition potential of LPS-induced NF-κB activation bycell permeable BODIPY-Ant-CC2 and BODIPY-Ant-LZ peptides, the presentinventors stably transfected the murine pre-B 70Z3 cell line withp12XlacZ-kB, which bears the β-galactosidase reporter gene under thecontrol of the NF-κB transcription factor. When the resulting cell line70Z3-C3 was treated for 5 hours with LPS (3 μg/ml) a 100 fold-activationof the LacZ gene was observed, indicating that our cellular assaymonitors NF-κB activation in response to LPS with extreme sensitivity(FIG. 3A, control “no peptide). Interestingly the incubation of cellswith 20 μM of both NEMO-derived polypeptides decreased significantly theNF-κB activation. This lowering was stronger in the presence ofBODIPY-Ant-LZ as compared to BODIPY-Ant-CC2. The inhibition effect wasessentially due to the NEMO sequence because the presence of theisolated and/or purified antennapedia peptide containing or notcontaining a N-terminal BODIPY label (BODIPY-Ant or Ant) induces thesame level of NF-κB activation as the control (FIG. 3A). Note that thebasal NF-κB activity measured in the absence of LPS was very similar inall samples indicating that both CC2 and LZ peptides abolish theresponsiveness to LPS without affecting the intrinsic basal NF-κBactivity. This was essential to minimize the in vivo cytotoxity,resulting mainly from apoptosis induced by inhibition of NF-κB (Chen,2003, Nature Med.).

To determine whether the BODIPY-Ant-LZ or the BODIPY-Ant-CC2 peptide isthe most efficient inhibitor, the present inventors next measured theconcentration dependent inhibition of each peptide. As shown in FIG. 3B,both NEMO peptides exhibit NF-κB dose dependant inhibition of NF-κB inresponse to LPS. BODIPY-Ant-LZ inhibited NF-κB to a greater extent thanBODIPY-Ant-CC2 did with IC₅₀ values of 3 μM and 22 μM respectively (FIG.3C). This striking difference could be explained by the NLM motifincluded in the LZ sequence. Consistent with the intracellular nature ofthe NEMO target, both LZ and CC2 peptides not fused to the antennapediaprotein transduction domain (PTD) exhibited the same level of activationas the control (FIG. 3D), confirming that NEMO derived peptides mustcross the cell membrane for inhibition of NF-κB. Taken together theseresults indicate that peptides that mimic the two coiled-coil sequencesof the NEMO oligomerization domain are potent peptide inhibitors ofNF-κB activation in response to LPS.

Mutations in the Hydrophobic Core of the LZ and CC2 Coiled-Coils Disrupttheir Specific Inhibition of the NF— B Signaling Pathway

Theoretically, If BODIPY-Ant-LZ or BODIPY-Ant-CC2 peptide inhibit NF-κBactivation through specific binding to the NEMO oligomerization domain,mutations that disrupt the coiled-coil association should thereforeexhibit impaired abilities to inhibit NF-κB inhibition. α-helicalcoiled-coil interactions have been extensively studied and most of therules governing their specific assembly have been well documented(Vinson, 2002, Mol. Cell. Biol.). The coiled-coil interface that isrepresented by the first (a) and fourth position (d) of the heptadrepeat is generally occupied by hydrophobic amino acids. Proline orglycine is largely excluded to preserve the helical architecture. Corepolar residues are destabilizing relative to leucine substitutions,especially when changes occur at d positions. Considering these rules,the present inventors synthesized a variant of BODIPY-Ant-LZ containingtwo mutations L→S at the d positions (BODIPY-Ant-LZ (Mu) and a variantof BODIPY-Ant-CC2 containing two mutations L→G and one mutation I→G atthe a positions (BODIPY-Ant-CC2 (Mu) (FIGS. 4A and 4B, and Table 1). Totest the effects of these mutations on the potential inhibition of NF-κBactivation, the present inventors developed a more stringent cellularassay that consists of the internalization of the peptides for 2 hoursfollowed by an extensive washing of 70Z3-C3 cells to remove anyremaining peptide in the extracellular media. Cells were allowed to growfor at least 24 hours before LPS-induced NF-κB activation. In this way,peptide interference with the receptor binding LPS was excluded. As withthe cellular assay described above, the BODIPY-Ant-CC2 (WT) and theBODIDY-Ant-LZ (WT) also inhibited NF-κB activation with a 1.7 and5.8-fold reduction respectively (FIGS. 4A and 4B) when used at a 10 μMconcentration. This indicates that the peptides do not competitively acton the receptor binding of LPS. As expected, the presence of the CC2variant (BODIPY-Ant-CC2 (Mu)) did not affect the NF-κB activation sinceβ-galactosidase activity was equivalent to that of the control (FIG. 4A,no peptide). In response to LPS, NF-κB is more strongly activated in thepresence of the BODIPY-Ant LZ mutant than in the presence of wild type.However, unlike the BODIPY-Ant-CC2 (Mu), a slight inhibition of the LZmutant was observed when compared to the control (15%). When takentogether, these data demonstrate that CC2 and LZ mutants are unable toinhibit the LPS-induced NF-κB activation as effectively the wild typedid.

Inhibition of NF-κB Activation is Mediated by a Specific Coiled CoilInteraction of the LZ Peptide.

Computational analyzes using the program MULTICOIL (Wolf, 1997, ProteinScience) predicted that greater than 5% of all putative ORFs found insequenced genomes are predicted to contain coiled-coil motifs (Newman,2000, Proc. Natl. Acad. Sci. USA) and that approximately 2-4% of aminoacids in proteins are estimated to adopt coiled-coil folds (Berger,1995, Proc. Natl. Acad. Sci. USA). This abundance raises the question ifthe NEMO derived-LZ peptide maintains its coiled-coil interactionpartnering specificity in vivo. To address this question, the presentinventors synthesized another coiled-coil peptide that mimics thesequence of the GCN4 leucine zipper and tested its ability to inhibitNF-κB activation. BODIPY-Ant-GCN4 contained the antennapedia sequence atits N-terminus and a short SKGMQ linker (SEQ ID NO: 9) identical to theCC2 sequence for convenience of peptide delivery (Table 1). It was alsolabeled at its N-terminus with BODIPY to monitor its cellular uptake byFACS (data not shown). The GCN4 peptide displays a low sequencesimilarity with the LZ sequence of NEMO (22%) but identical residues aremostly represented by leucines at d positions (FIG. 5). These residuescontribute most of the energy to coiled-coil oligomerization stability(Vinson, 2002, Mol. Cell. Biol.). Note that a positions which areimportant for coiled-coil specificity (Vinson, 2002, Mol. Cell. Biol)are composed of a set of different amino acids. While GCN4 is composedof hydrophobic residues and the typical asparagine residue, the LZ ofNEMO contains two charged amino acids R and K (FIG. 4B and FIG. 5A).Thus, these residues, which are located at the coiled coil interfacelikely, contributes to the selectivity of coiled coil interaction.

FIG. 5B shows the effect of the BODIPY-Ant-GCN4 at a 10 μM concentrationon the inhibition of NF-κB activation in response to LPS. To compare theeffects of coiled coil sequences, the present inventors used thestringent cellular assay described above. BODIPY-Ant-GCN4, unlikeBODIPY-Ant-LZ, has no ability to inhibit NF-κB activation since thelevel of NF-κB activation was near that of the control without peptide.Taken together, these results strongly support the hypothesis that theLZ peptide of NEMO inhibits NF-κB activation through selectivecoiled-coil interactions.

The Antennapedia Sequence Induces Monomerization of NF-κB PeptidicInhibitors

The antennapedia sequence is a protein transduction domain (PTD) whichadopts an alpha-helical amphiphatic structure (Prochiantz, 2000, Curr.Opin. Cell Biol.). When fused to the N terminus of a coiled-coilsequence like CC2 or LZ, the antennapedia could alter the coiled-coilassociation by covering the hydrophobic interface of the coiled-coilthrough intramolecular interactions. To examine the effect of N-fusionof the antennapedia peptide on the oligomerization properties of the CC2and the LZ peptides, the present inventors analyzed peptides containingor not containing the antennapedia sequence at their N-terminus by gelfiltration. As shown in FIG. 6, all peptides containing an N-terminalfusion of antennapedia co-elute with an elution volume corresponding totheir monomeric forms as compared to globular protein markers. Note thatthe present inventors had to add a detergent in the buffer below its cmcto improve peptide recoveries. When injected at the same 10 μMconcentration, CC2 wild type and LZ wild type without the antennapediaN-fusion oligomerize. CC2 (WT) forms a trimer whereas LZ (WT) forms adimer as recently reported (Traincard et al). As expected, when a CC2mutant was chemically obtained with three of its aliphatic residues atpositions replaced with glycine residues, it lost its ability tooligomerize (bottom panel, dashed line). The effect of the two L→Smutations at d positions was less strong with LZ (mutant). However, thepresent inventors still detected dimerization of the LZ mutant at a 10μM concentration (dashed line) although its association was markedlyreduced by mutations as compared to the wild type LZ (solid line). Takentogether, these data indicate that the N-fusion of the antennapediasequence to both CC2 and LZ peptides alter homotypic coiled-coilinteractions, facilitating the monomerization of the NEMOderived-peptides. Furthermore these results also show that residuechanges at a and d position alter oligomerization of LZ and CC2peptides. Thus, it is likely that the synthetic peptides form α-helicalcoiled-coil structures.

Homo- and Heterotypic Interactions of CC2 and LZ Peptides with andwithout the N-Fusion of Antennapedia Sequence.

Because the N-fusion of antennapedia modifies the oligomerizationproperties of the CC2 and LZ peptides, the present inventors nextstudied by fluorescence polarization whether the Ant-CC2 and the Ant-LZmonomers labeled with BODIPY could bind to the NEMO-derived polypeptidesdevoid of the antennapedia sequence. These peptides CC2 and LZ may bealso considered as the in vivo binding target for both cell permeableBODIPY-Ant-CC2 and BODIPY-Ant-LZ NF-κB inhibitors. FIG. 7 shows atypical binding isotherm for the interaction of various concentrationsof the CC2 peptide with a fixed concentration of the BODIPY-Ant-CC2. Theshape of the binding curve is not sigmoidal, indicating that CC2 bindsto the BODIPY-Ant-CC2 peptide without cooperativity. The stoichiometrycalculated from the intercept between the tangent of the initial part ofthe anisotropy and the asymptote is equal to 0.8. Taking into accountthis stoichiometry, the dissociation constant K_(D) is 15.2 μM. Similarresults were obtained when a fixed concentration of the BODIPY-Ant-LZwas titrated with various concentrations of the CC2 peptide as thepresent inventors previously reported (Inset FIG. 7, Traincard et al.).Collectively, these data demonstrate that both Ant-CC2 and Ant-LZmonomers binds in vitro to the CC2 peptide composing the minimaloligomerization domain of NEMO.

Cell Death in Human Retinoblastoma Cell is Induced by NF-κB InhibitorsAnt-CC2 and Ant-LZ, But not by their Mutants Ant-CC2 (Mu) and Ant-LZ(Mu)

It has become clear that constitutively activated NF-κB transcriptionfactors have been associated with several aspects of tumorigenesis(Karin review), including most of six essential alterations in cellphysiology that dictate the conversion of normal human cells into cancercells (Hanahan, 2000, Cell for review). This led to a significantenthusiasm for the use of NF-κB inhibitors as a new anti-cancer therapy.Promising results have been reported recently using proteasomeinhibitors or the SN50 peptide that blocks the nuclear translocation(Orlowski, 2002, Trends in Molecular Medicine; Mitsiades, 2002, Blood).However, the specificity of these agents on NF-κB inhibition have beenquestioned. Poulaki et al. (2002, Am J. Pathol.) showed recently thatthe treatment of the human retinoblastoma (Rb) cell lines Y79 with SN50peptide induced apoptosis of cancer cells. Sequence alignment of murineand human NEMO proteins indicate that the minimal oligomerization domainof NEMO is strictly conserved, suggesting that similar effects of NF-κBinhibition could be observed in rodent as well as in human cells.

Given that specific NF-κB inhibition may trigger apoptosis of cancercells, the present inventors examined the effects of both cell-permeableAnt-LZ and Ant-CC2 peptides on human retinoblastoma cell viability. Inthese experiments, the present inventors used NEMO-derived polypeptideswithout a N-terminal BODIPY labeling to prevent any interference withthe MTS assay (see “Materials and Methods”). As shown in FIG. 7, thepresent inventors found a dose dependence of the Y79 cell viability whencells were treated for 3 hours with Ant-CC2 (FIG. 8A) or Ant-LZ (FIG.8B). The effect of the Ant-LZ peptide on cell death was stronger thanthat of the Ant-CC2. This induction of cell death was significant sinceRb cell survival was 20% and 65% with the Ant-LZ and the Ant-CC2peptides respectively when cancer cells were treated for 3 hours at a 20mM concentration. Remarkably, the same cell treatment with the Ant-CC2(Mu) (FIG. 8A) or with the Ant-CC2 (Mu) (FIG. 8B) did not induceconcentration cell death as did WT peptides. These effects on cell deathwere essentially due to the NEMO sequence because a longer treatment ofY79 cell lines with the antennapedia peptide did not affect cellsurvival (FIG. 8C). In contrast, 80% and 55% of Y79 cell died in thepresence of Ant-LZ and Ant-CC2 respectively at 5 μM concentration (FIG.8C). Taken together, these results indicate that specific NF-κBinhibition by Ant-CC2 and Ant-LZ peptides induce cell death in Rb celllines, validating the use of specific NF-κB inhibitors as anticancerchemotherapy.

In the absence of the pro-inflammatory signals, all peptides assayedhave no detectable cytoxicity for lymphocytes B tested at aconcentration up to 30 μM according to the MTS assay, the directobservation under microscope, and the forward scatter-FSC and sidescatter-SSC parameters deduced from FACS analyses. However, the presentinventors could detect a slight cell death by FACS in aconcentration-dependent manner when LPS stimulated the pre-B lymphocytesin the presence of NEMO-derived polypeptides. The cellular proportion ofcell death was 9% in the absence of stimuli at a 20 μM concentration ofAnt-CC2 whereas it increased at 13% after LPS stimulation (data notshown). This was in agreement with the role of the NF-κB pathway inprotecting cells from apoptosis. The cell death was more pronounced andfast on the Rb cell lines Y79 in which constitutive NF-κB activity hasbeen reported (Poulaki, 2002, Am J. Pathol.). The present inventors didnot demonstrate here by the Annexin V labeling and the TUNEL method thatthe NEMO peptides-induced cell death is indeed apoptosis. Neverthelessconsidering the role of the NF-κB pathway in the regulation ofapoptosis, it is likely that the cell death induced by NEMO-derivedpolypeptides is apoptotic in nature.

The results set forth above, regardless of the nature of the futureNF-κB inhibitors (organic or peptidomimetic compounds), targeting NEMO'soligomerization will remain a more attractive and promising strategy ascompared to those of IKK kinase activity and of NEMO-kinase associationbecause this molecular event strictly depend on the pro-inflammatorysignal. Therefore, such drugs would interfere less with the basal NF-κBactivity in normal cells that is required for cell viability.

Peptides Derived from the N-Terminal Region of Wild-Type NEMO

The present inventors explored the N-terminal region of the wild-typeNEMO, in particular the NLM conserved motif (residues 293-322 of SEQ IDNO: 12) appearing in FIG. 1A. To this end, the following sequences wereproduced (see Table 1 for the corresponding sequence):

NLM-DR (SEQ ID NO: 30) Ant.NLM-DR (SEQ ID NO: 31) Tat NLM-DR (SEQ ID NO:32) R7-NLM-DR (SEQ ID NO: 33) R9-NLM-DR (SEQ ID NO: 34)

NLM-DR is a 21 amino acid “motif” (and the corresponding wild type NLMcovering the same amino acid range) derived from the larger 30 aminoacid conserved NLM motif set forth in FIG. 1A. The NLM-DR has beenmutated from the wild type NLM sequence in that the aspartic acid atresidue 11 in the wild type sequence has been replaced by an arginine(see Table 1 and SEQ ID NO: 30).

The present inventors compared NLM-DR to the corresponding NLM peptideby measuring the respective cooperativity indices, which evidence anadvantage of NLM-DR mutated peptide over the wild-type.

The calculation of the “cooperativity indice” by means of Hillcoefficient is based on the following formula

Boundmax×L^(n)/(KD ^(n)+L^(n))

Wherein

-   -   Boundmax is the maximum concentration of the bound ligand,    -   L is the concentration of the free ligand,    -   n is the cooperativity indice and    -   KD, is the affinity constant of the ligand by the protein.

The cooperative indices calculated by means of the Hill coefficientcalculation are:

-   -   For the mutant NLM peptide (NLM-DR)—a dissociation constant Kd        of 170 μM and a cooperativity indice of 1.4 (1326 μM); and    -   For the corresponding wild-type NLM peptide (NLM)—a dissociation        constant Kd of 240 μM and a cooperativity indice of 2.1 (99642        μM).

If these values were to be compared to those obtained from the curveswithout accounting for cooperativity, the affinity of NLM-DR peptidewould be 75 times higher than the affinity of wild-type NLM peptide.

The present inventors also evaluated the biologically relevant resultsfor the aforementioned forms of NLM-DR as they apply to the inhibitionof NF-κB activation pathway, IC50 and toxicity results (FACS). Theresults are presented in Table 2:

TABLE 2 Properties of NLM-DR derived peptides Cytotoxicity* Cellularuptake MTS Cell morphology relative efficiency Peptide Assay FACSMicroscope (FACS) IC 50 BODIPY-Ant-NLM-DR no + + + 1.1 μMBODIPY-TAT-NLM-DR no no no + 1 μM BODIPY-R9-NLM-DR no no no ++ 0.9 μMBODIPY-R7-NLM-DR n.d. no no ++++ 0.9 μM *Cytotoxicity analysed by theindicated technique is scaled from no toxicity (no) to high toxicity(++++); the MTS cell proliferation assay was from Promega; n.d. = notdetermined.

Numerous modifications and variations on the present invention arepossible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the accompanying claims, theinvention may be practiced otherwise than as specifically describedherein.

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1: A purified polynucleotide which encodes a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO:
 7. 2: The purifiedpolynucleotide of claim 1, wherein said polypeptide inhibits the NF-κBpathway. 3: The purified polynucleotide of claim 2, wherein saidpolypeptide disrupts NEMO oligomerization. 4: The purifiedpolynucleotide of claim 1, wherein said amino acid sequence is SEQ IDNO:
 2. 5: The purified polynucleotide of claim 1, wherein said aminoacid sequence is SEQ ID NO:
 3. 6: The purified polynucleotide of claim1, wherein said amino acid sequence is SEQ ID NO:
 6. 7: The purifiedpolynucleotide of claim 1, wherein said amino acid sequence is SEQ IDNO:
 7. 8: A purified polynucleotide which is complementary to thepolynucleotide of claim
 1. 9: A purified polynucleotide which is atleast 70% identical to the polynucleotide of claim
 1. 10: A purifiedpolynucleotide which is at least 80% identical to the polynucleotide ofclaim
 1. 11: A purified polynucleotide which is at least 90% identicalto the polynucleotide of claim
 1. 12: A purified polynucleotide whichhybridizes under stringent conditions to the polynucleotide of claim 1;wherein said stringent conditions comprise washing in 5×SSC at atemperature from 50 to 68° C. 13: A vector comprising the purifiedpolynucleotide of claim
 1. 14: A host cell comprising the purifiedpolynucleotide of claim
 1. 15: A purified polypeptide comprising anamino acid sequence that is at least 70% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 3 and SEQ IDNO:
 7. 16: The polypeptide of claim 15, wherein the amino acid sequenceis SEQ ID NO:
 7. 17: A purified polypeptide which is at least 80%identical to the polypeptide of claim
 15. 18: A purified polypeptidewhich is at least 90% identical to the polypeptide of claim
 15. 19: Apurified polypeptide which is at least 95% identical to the polypeptideof claim
 15. 20: The purified polypeptide of claim 15, wherein saidpolypeptide inhibits the NF-κB pathway. 21: The purified polypeptide ofclaim 20, wherein said polypeptide disrupts NEMO oligomerization. 22: Apolypeptide fusion construct comprising an amino acid sequence that isat least 70% identical to the amino acid sequence of SEQ ID NO: 7, whichis linked to a polypeptide having a high transduction potential, whereinsaid polypeptide fusion construct inhibits the NF-κB pathway. 23: Thepolypeptide of claim 22, wherein the amino acid sequence is at least 80%identical to SEQ ID NO:
 7. 24: The polypeptide of claim 22, wherein theamino acid sequence is at least 90% identical to SEQ ID NO:
 7. 25: Thepolypeptide of claim 22, wherein the amino acid sequence is at least 95%identical to SEQ ID NO:
 7. 26: The polypeptide of claim 22, wherein saidpolypeptide fusion construct disrupts NEMO oligomerization. 27: Thepolypeptide of claim 22, wherein said linked is by an amino acid spacersequence having a length ranging from 1-35 amino acids. 28: Thepolypeptide of claim 27, wherein said amino acid spacer sequence isselected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 41.29: The polypeptide of claim 22, wherein said polypeptide having a hightransduction potential has an amino acid sequence of SEQ ID NO:
 1. 30:The polypeptide of claim 29, wherein the polypeptide fusion constructhas the amino acid sequence of SEQ ID NO:
 6. 31: A method of inhibitingthe NF-κB signaling pathway comprising contacting an eukaryotic cellwith a polypeptide fusion construct comprising an amino acid sequencethat is at least 70% identical to the amino acid sequence selected fromthe group consisting of SEQ ID NO: 3 and SEQ ID NO: 7, which is linkedto a polypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway. 32: The methodof claim 31, wherein said polypeptide fusion construct has the aminoacid sequence of SEQ ID NO:
 2. 33: The method of claim 31, wherein saidpolypeptide fusion construct has the amino acid sequence of SEQ ID NO:6. 34: A method of disrupting NEMO oligomerization comprising contactingsaid NEMO with a polypeptide fusion construct comprising an amino acidsequence that is at least 70% identical to the amino acid sequenceselected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 7,which is linked to a polypeptide having a high transduction potential,wherein said polypeptide fusion construct inhibits the NF-κB pathway.35: The method of claim 34, wherein said polypeptide fusion constructhas the amino acid sequence of SEQ ID NO:
 2. 36: The method of claim 34,wherein said polypeptide fusion construct has the amino acid sequence ofSEQ ID NO:
 6. 37: A method of modulating or treatment of a disorderregulated by the NF-κB signaling pathway in a subject in need thereofcomprising administering an effective amount of a composition comprisinga polypeptide fusion construct comprising an amino acid sequence that isat least 70% identical to the amino acid sequence selected from thegroup consisting of SEQ ID NO: 3 and SEQ ID NO: 7, which is linked to apolypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway, and one or morepharmaceutically acceptable carriers or excipients. 38: The method ofclaim 37, wherein said polypeptide fusion construct has the amino acidsequence of SEQ ID NO:
 2. 39: The method of claim 37, wherein saidpolypeptide fusion construct has the amino acid sequence of SEQ ID NO:6. 40: The method of claim 37, wherein said subject in need thereof is ahuman. 41: The method of claim 37, wherein said effective amount rangesfrom 0.1 mg/Kg/day to 30 mg/Kg/day. 42: The method of claim 37, whereinsaid disorder regulated by the NF-κB signaling pathway is selected fromthe group consisting of inflammatory responses, oncogenesis, and viralinfection. 43: The method of claim 37, wherein said administering isselected from the group consisting of orally, rectally, nasally,parenterally, intracisternally, intravaginally, intraperitoneally,sublingually, topically, and bucally. 44: The method of claim 37,wherein said administering is intravenously. 45: A method of regulatingcell proliferation or apoptosis in a subject in need thereof comprisingadministering an effective amount of a composition comprising apolypeptide fusion construct comprising an amino acid sequence that isat least 70% identical to the amino acid sequence selected from thegroup consisting of SEQ ID NO: 3 and SEQ ID NO: 7, which is linked to apolypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway, and one or morepharmaceutically acceptable carriers or excipients. 46: The method ofclaim 45, wherein said polypeptide fusion construct has the amino acidsequence of SEQ ID NO:
 2. 47: The method of claim 45, wherein saidpolypeptide fusion construct has the amino acid sequence of SEQ ID NO:6. 48: The method of claim 45, wherein said subject in need thereof is ahuman. 49: The method of claim 45, wherein said effective amount rangesfrom 0.1 mg/Kg/day to 30 mg/Kg/day. 50: The method of claim 45, whereinsaid administering is selected from the group consisting of orally,rectally, nasally, parenterally, intracisternally, intravaginally,intraperitoneally, sublingually, topically, and bucally. 51: The methodof claim 45, wherein said administering is intravenously. 52: A methodof regulating B or T lymphocytes in antigenic stimulation in a subjectin need thereof comprising administering an effective amount of acomposition comprising a polypeptide fusion construct comprising anamino acid sequence that is at least 70% identical to the amino acidsequence selected from the group consisting of SEQ ID NO: 3 and SEQ IDNO: 7, which is linked to a polypeptide having a high transductionpotential, wherein said polypeptide fusion construct inhibits the NF-κBpathway, and one or more pharmaceutically acceptable carriers orexcipients. 53: The method of claim 52, wherein said polypeptide fusionconstruct has the amino acid sequence of SEQ ID NO:
 2. 54: The method ofclaim 52, wherein said polypeptide fusion construct has the amino acidsequence of SEQ ID NO:
 6. 55: The method of claim 52, wherein saidsubject in need thereof is a human. 56: The method of claim 52, whereinsaid effective amount ranges from 0.1 mg/Kg/day to 30 mg/Kg/day. 57: Themethod of claim 52, wherein said administering is selected from thegroup consisting of orally, rectally, nasally, parenterally,intracisternally, intravaginally, intraperitoneally, sublingually,topically, and bucally. 58: The method of claim 52, wherein saidadministering is intravenously. 59: A method of identifying polypeptidesthat modulate oligomerization of NEMO comprising a) identifying acandidate polypeptide sequence; b) creating a polypeptide fusionconstruct by linking said candidate polypeptide sequence to apolypeptide having a high transduction potential via a spacer sequence;c) contacting a cell culture with the polypeptide fusion construct; andd) monitoring the activity of the NF-κB signaling pathway; e) comparingthe activity of the NF-κB signaling pathway in the presence of saidpolypeptide fusion construct to the activity of the NF-κB signalingpathway in the absence of said polypeptide fusion construct to determinethe relative inhibition by said polypeptide fusion construct; and f)correlating relative inhibition by said polypeptide fusion construct toNEMO oligomerization. 60: The method of claim 59, wherein said candidatepolypeptide sequence has a coiled-coil or helical structure. 61: Themethod of claim 60, wherein said candidate polypeptide sequence has20-60 amino acids. 62: The method of claim 59, wherein said candidatepolypeptide sequence is derived from NEMO. 63: The method of claim 59,wherein said spacer sequence has a length ranging from 1-35 amino acids.64: The method of claim 63, wherein said spacer sequence is selectedfrom the group consisting of SEQ ID NO: 40 and SEQ ID NO:
 41. 65: Themethod of claim 59, wherein said polypeptide having a high transductionpotential has an amino acid sequence of SEQ ID NO:
 1. 66: The method ofclaim 59, wherein said cell culture comprises pre-B 70Z/3 lymphocytesthat have been transfected with NF-κB dependent β-glactosidase reportergene. 67: The method of claim 59, wherein said polypeptide fusionconstruct further comprises an N-terminal cysteine residue. 68: Themethod claim 65, further comprising b-1) labeling said polypeptidefusion construct; and c-1) monitoring cellular uptake of the labeledpolypeptide fusion construct. 69: The method of claim 68, wherein saidlabeling comprises chemically reacting the cysteine residue with afluorophore. 70: The method of claim 69, wherein said fluorophore isBODIPY. 71: The method of claim 68, wherein said monitoring cellularuptake is by FACS. 72: An purified polynucleotide which encodes apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ IDNO:
 16. 73: The purified polynucleotide of claim 72, wherein saidpolypeptide inhibits the NF-κB pathway. 74: The purified polynucleotideof claim 73, wherein said polypeptide disrupts NEMO oligomerization. 75:The purified polynucleotide of claim 72, wherein said amino acidsequence is SEQ ID NO:
 13. 76: The purified polynucleotide of claim 72,wherein said amino acid sequence is SEQ ID NO:
 14. 77: The purifiedpolynucleotide of claim 72, wherein said amino acid sequence is SEQ IDNO:
 15. 78: The purified polynucleotide of claim 72, wherein said aminoacid sequence is SEQ ID NO:
 16. 79: An purified polynucleotide which iscomplementary to the polynucleotide of claim
 72. 80: An purifiedpolynucleotide which is at least 70% identical to the polynucleotide ofclaim
 72. 81: An purified polynucleotide which is at least 80% identicalto the polynucleotide of claim
 72. 82: An purified polynucleotide whichis at least 90% identical to the polynucleotide of claim
 72. 83: Anpurified polynucleotide which hybridizes under stringent conditions tothe polynucleotide of claim 72; wherein said stringent conditionscomprise washing in 5×SSC at a temperature from 50 to 68° C. 84: Avector comprising the purified polynucleotide of claim
 72. 85: A hostcell comprising the purified polynucleotide of claim
 72. 86: A purifiedpolypeptide comprising an amino acid sequence that is at least 70%identical to the amino acid sequence of SEQ ID NO:
 16. 87: A purifiedpolypeptide which is at least 80% identical to the polypeptide of claim86. 88: An purified polypeptide which is at least 90% identical to thepolypeptide of claim
 86. 89: An purified polypeptide which is at least95% identical to the polypeptide of claim
 86. 90: The purifiedpolypeptide of claim 86, wherein said polypeptide inhibits the NF-κBpathway. 91: The purified polypeptide of claim 90, wherein saidpolypeptide disrupts NEMO oligomerization. 92: A polypeptide fusionconstruct comprising an amino acid sequence that is at least 70%identical to the amino acid sequence of SEQ ID NO: 16, which is linkedto a polypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway. 93: Thepolypeptide of claim 92, wherein the amino acid sequence is at least 80%identical to SEQ ID NO:
 16. 94: The polypeptide of claim 92, wherein theamino acid sequence is at least 90% identical to SEQ ID NO:
 16. 95: Thepolypeptide of claim 92, wherein the amino acid sequence is at least 95%identical to SEQ ID NO:
 16. 96: The polypeptide of claim 92, whereinsaid polypeptide fusion construct disrupts NEMO oligomerization. 97: Thepolypeptide of claim 92, wherein said linked is by an amino acid spacersequence having a length ranging from 1-35 amino acids. 98: Thepolypeptide of claim 97, wherein said amino acid spacer sequence isselected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 41.99: The polypeptide of claim 92, wherein said polypeptide having a hightransduction potential has an amino acid sequence of SEQ ID NO:
 1. 100:The polypeptide of claim 99, wherein the polypeptide fusion constructhas the amino acid sequence of SEQ ID NO:
 15. 101: A method ofinhibiting the NF-κB signaling pathway comprising contacting aneukaryotic cell with a polypeptide fusion construct comprising an aminoacid sequence that is at least 70% identical to the amino acid sequenceselected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16,which is linked to a polypeptide having a high transduction potential,wherein said polypeptide fusion construct inhibits the NF-κB pathway.102: The method of claim 101, wherein said polypeptide fusion constructhas the amino acid sequence of SEQ ID NO:
 13. 103: The method of claim101, wherein said polypeptide fusion construct has the amino acidsequence of SEQ ID NO:
 15. 104: A method of disrupting NEMOoligomerization comprising contacting said NEMO with a polypeptidefusion construct comprising an amino acid sequence that is at least 70%identical to the amino acid sequence selected from the group consistingof SEQ ID NO: 14 and SEQ ID NO: 16, which is linked to a polypeptidehaving a high transduction potential, wherein said polypeptide fusionconstruct inhibits the NF-κB pathway. 105: The method of claim 104,wherein said polypeptide fusion construct has the amino acid sequence ofSEQ ID NO:
 13. 106: The method of claim 104, wherein said polypeptidefusion construct has the amino acid sequence of SEQ ID NO:
 15. 107: Amethod of modulating or treatment of a disorder regulated by the NF-κBsignaling pathway in a subject in need thereof comprising administeringan effective amount of a composition comprising a polypeptide fusionconstruct comprising an amino acid sequence that is at least 70%identical to the amino acid sequence selected from the group consistingof SEQ ID NO: 3 and SEQ ID NO: 7, which is linked to a polypeptidehaving a high transduction potential, wherein said polypeptide fusionconstruct inhibits the NF-κB pathway, and one or more pharmaceuticallyacceptable carriers or excipients. 108: The method of claim 107, whereinsaid polypeptide fusion construct has the amino acid sequence of SEQ IDNO:
 13. 109: The method of claim 107, wherein said polypeptide fusionconstruct has the amino acid sequence of SEQ ID NO:
 15. 110. The methodof claim 107, wherein said subject in need thereof is a human. 111: Themethod of claim 107, wherein said effective amount ranges from 0.1mg/Kg/day to 30 mg/Kg/day. 112: The method of claim 107, wherein saiddisorder regulated by the NF-κB signaling pathway is selected from thegroup consisting of inflammatory responses, oncogenesis, and viralinfection. 113: The method of claim 107, wherein said administering isselected from the group consisting of orally, rectally, nasally,parenterally, intracisternally, intravaginally, intraperitoneally,sublingually, topically, and bucally. 114: The method of claim 107,wherein said administering is intravenously. 115: A method of regulatingcell proliferation or apoptosis in a subject in need thereof comprisingadministering an effective amount of a composition comprising apolypeptide fusion construct comprising an amino acid sequence that isat least 70% identical to the amino acid sequence selected from thegroup consisting of SEQ ID NO: 14 and SEQ ID NO: 16, which is linked toa polypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway, and one or morepharmaceutically acceptable carriers or excipients. 116: The method ofclaim 115, wherein said polypeptide fusion construct has the amino acidsequence of SEQ ID NO:
 13. 117. The method of claim 115, wherein saidpolypeptide fusion construct has the amino acid sequence of SEQ ID NO:15.
 118. The method of claim 115, wherein said subject in need thereofis a human.
 119. The method of claim 115, wherein said effective amountranges from 0.1 mg/Kg/day to 30 mg/Kg/day. 120: The method of claim 115,wherein said administering is selected from the group consisting oforally, rectally, nasally, parenterally, intracisternally,intravaginally, intraperitoneally, sublingually, topically, and bucally.121: The method of claim 115, wherein said administering isintravenously. 122: A method of regulating B or T lymphocytes inantigenic stimulation in a subject in need thereof comprisingadministering an effective amount of a composition comprising apolypeptide fusion construct comprising an amino acid sequence that isat least 70% identical to the amino acid sequence selected from thegroup consisting of SEQ ID NO: 14 and SEQ ID NO: 16, which is linked toa polypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway, and one or morepharmaceutically acceptable carriers or excipients. 123: The method ofclaim 122, wherein said polypeptide fusion construct has the amino acidsequence of SEQ ID NO:
 13. 124: The method of claim 122, wherein saidpolypeptide fusion construct has the amino acid sequence of SEQ ID NO:15. 125: The method of claim 122, wherein said subject in need thereofis a human. 126: The method of claim 122, wherein said effective amountranges from 0.1 mg/Kg/day to 30 mg/Kg/day. 127: The method of claim 122,wherein said administering is selected from the group consisting oforally, rectally, nasally, parenterally, intracisternally,intravaginally, intraperitoneally, sublingually, topically, and bucally.128: The method of claim 122, wherein said administering isintravenously. 129: An purified polynucleotide which encodes apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, and SEQ ID NO:
 39. 130: The purified polynucleotide of claim129, wherein said polypeptide inhibits the NF-κB pathway. 131: Thepurified polynucleotide of claim 130, wherein said polypeptide disruptsNEMO oligomerization. 132: A purified polynucleotide which iscomplementary to the polynucleotide of claim
 129. 133: A purifiedpolynucleotide which is at least 70% identical to the polynucleotide ofclaim
 129. 134: A purified polynucleotide which is at least 80%identical to the polynucleotide of claim
 129. 135: A purifiedpolynucleotide which is at least 90% identical to the polynucleotide ofclaim
 129. 136: A purified polynucleotide which hybridizes understringent conditions to the polynucleotide of claim 129; wherein saidstringent conditions comprise washing in 5×SSC at a temperature from 50to 68° C. 137: A vector comprising the purified polynucleotide of claim129. 138: A host cell comprising the purified polynucleotide of claim129. 139: A purified polypeptide comprising an amino acid sequence thatis at least 70% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ IDNO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQID NO: 38, and SEQ ID NO:
 39. 140: A purified polypeptide which is atleast 80% identical to the polypeptide of claim
 139. 141: A purifiedpolypeptide which is at least 90% identical to the polypeptide of claim139. 142: A purified polypeptide which is at least 95% identical to thepolypeptide of claim
 139. 143: The purified polypeptide of claim 139,wherein said polypeptide inhibits the NF-κB pathway. 144: The purifiedpolypeptide of claim 143, wherein said polypeptide disrupts NEMOoligomerization. 145: A polypeptide fusion construct comprising an aminoacid sequence that is at least 70% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, which is linked to apolypeptide having a high transduction potential, wherein saidpolypeptide fusion construct inhibits the NF-κB pathway. 146: Thepolypeptide of claim 145, wherein the amino acid sequence is at least80% identical to said amino acid sequence. 147: The polypeptide of claim145, wherein the amino acid sequence is at least 90% identical to saidamino acid sequence. 148: The polypeptide of claim 145, wherein theamino acid sequence is at least 95% identical to said amino acidsequence. 149: The polypeptide of claim 145, wherein said polypeptidefusion construct disrupts NEMO oligomerization. 150: The polypeptide ofclaim 145, wherein said linked is by an amino acid spacer sequencehaving a length ranging from 1-35 amino acids. 151: The polypeptide ofclaim 150, wherein said amino acid spacer sequence is selected from thegroup consisting of SEQ ID NO: 40 and SEQ ID NO: 41 SEQ ID NO: 9 and SEQID NO:
 10. 152: The polypeptide of claim 145, wherein said polypeptidehaving a high transduction potential has an amino acid sequence of SEQID NO:
 1. 153: A method of inhibiting the NF-κB signaling pathwaycomprising contacting an eukaryotic cell with a polypeptide fusionconstruct of claim
 145. 154: A method of disrupting NEMO oligomerizationcomprising contacting said NEMO with a polypeptide fusion construct ofclaim
 145. 155: A method of modulating or treatment of a disorderregulated by the NF-κB signaling pathway in a subject in need thereofcomprising administering an effective amount of a composition comprisinga polypeptide fusion construct of claim 145 and one or morepharmaceutically acceptable carriers or excipients. 156: The method ofclaim 155, wherein said subject in need thereof is a human. 157: Themethod of claim 155, wherein said effective amount ranges from 0.1mg/Kg/day to 30 mg/Kg/day. 158: The method of claim 155, wherein saiddisorder regulated by the NF-κB signaling pathway is selected from thegroup consisting of inflammatory responses, oncogenesis, and viralinfection. 159: The method of claim 155, wherein said administering isselected from the group consisting of orally, rectally, nasally,parenterally, intracisternally, intravaginally, intraperitoneally,sublingually, topically, and bucally. 160: The method of claim 155,wherein said administering is intravenously. 161: A method of regulatingcell proliferation or apoptosis in a subject in need thereof comprisingadministering an effective amount of a composition comprising apolypeptide fusion construct of claim 145 and one or morepharmaceutically acceptable carriers or excipients. 162: The method ofclaim 161, wherein said subject in need thereof is a human. 163: Themethod of claim 161, wherein said effective amount ranges from 0.1mg/Kg/day to 30 mg/Kg/day. 164: The method of claim 161, wherein saidadministering is selected from the group consisting of orally, rectally,nasally, parenterally, intracisternally, intravaginally,intraperitoneally, sublingually, topically, and bucally. 165: The methodof claim 161, wherein said administering is intravenously. 166: A methodof regulating B or T lymphocytes in antigenic stimulation in a subjectin need thereof comprising administering an effective amount of acomposition comprising a polypeptide fusion construct of claim 145 andone or more pharmaceutically acceptable carriers or excipients. 167: Themethod of claim 166, wherein said subject in need thereof is a human.168: The method of claim 166, wherein said effective amount ranges from0.1 mg/Kg/day to 30 mg/Kg/day. 169: The method of claim 166, whereinsaid administering is selected from the group consisting of orally,rectally, nasally, parenterally, intracistemally, intravaginally,intraperitoneally, sublingually, topically, and bucally. 170: The methodof claim 166, wherein said administering is intravenously.